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'description' => '<p>5<span>-hmC is a DNA modification which results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. Preliminary results indicate that 5-hmC may have important roles distinct from 5-methylcytosine (5-mC). Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics.</span></p>',
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<p><strong>Figure 1. Hydroxymethylated DNA IP results obtained with our hMeDIP kit (Cat. No. AF-104-0016)</strong><br /> Hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit. This kit includes: the monoclonal antibody against 5-hydroxymethylcytosine (Cat. No. MAb-633HMC-050), 5-hmC, 5-mC & cytosine DNA standards & Rat IgG (Cat. No. AF-105-0025). The DNA was prepared with the GenDNA module and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. 1 μg of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP’d material has been analysed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control • with Rat IgG as negative control (0.06%, almost no recovery) • with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control • with Rat IgG as negative control (0.03%, almost no recovery) • with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control • with Rat IgG as negative control (0.04%, almost no recovery) • with 5-hmC (97.60% recovery, almost full recovery) These results clearly demonstrate the high specificity and efficiency of the 5-hydroxymethylcytosine monoclonal antibody.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig2.png" alt="ELISA" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 2. Determination of the 5-hmC rat monoclonal antibody titer</strong><br /> To determine the titer, an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody directed against 5-hmC (Cat No. MAb-633HMC-050, MAb-633HMC-100) in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig3.png" alt="Dot blot" style="display: block; margin-left: auto; margin-right: auto;" width="200" height="200" /></p>
<p><strong>Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls</strong><br /> Figure 3A: Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig4.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls</strong><br /> 200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode « 5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.</p>',
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Preliminary results indicate that 5-hmC may have important roles distinct from 5-mC. Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics : 5-hydroxymethylcytosine may well represent a new pathway to demethylate DNA involving a repair mechanism converting 5-hmC to cytosine and, as such open up entirely new perspectives in epigenetic studies.
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'description' => '<p>5<span>-hmC is a DNA modification which results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. Preliminary results indicate that 5-hmC may have important roles distinct from 5-methylcytosine (5-mC). Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics.</span></p>',
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<p><strong>Figure 1. Hydroxymethylated DNA IP results obtained with our hMeDIP kit (Cat. No. AF-104-0016)</strong><br /> Hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit. This kit includes: the monoclonal antibody against 5-hydroxymethylcytosine (Cat. No. MAb-633HMC-050), 5-hmC, 5-mC & cytosine DNA standards & Rat IgG (Cat. No. AF-105-0025). The DNA was prepared with the GenDNA module and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. 1 μg of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP’d material has been analysed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control • with Rat IgG as negative control (0.06%, almost no recovery) • with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control • with Rat IgG as negative control (0.03%, almost no recovery) • with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control • with Rat IgG as negative control (0.04%, almost no recovery) • with 5-hmC (97.60% recovery, almost full recovery) These results clearly demonstrate the high specificity and efficiency of the 5-hydroxymethylcytosine monoclonal antibody.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig2.png" alt="ELISA" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 2. Determination of the 5-hmC rat monoclonal antibody titer</strong><br /> To determine the titer, an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody directed against 5-hmC (Cat No. MAb-633HMC-050, MAb-633HMC-100) in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig3.png" alt="Dot blot" style="display: block; margin-left: auto; margin-right: auto;" width="200" height="200" /></p>
<p><strong>Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls</strong><br /> Figure 3A: Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig4.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls</strong><br /> 200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode « 5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.</p>',
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<p>Preliminary results indicate that 5-hmC may have important roles distinct from 5-mC. Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics : 5-hydroxymethylcytosine may well represent a new pathway to demethylate DNA involving a repair mechanism converting 5-hmC to cytosine and, as such open up entirely new perspectives in epigenetic studies.</p>
<p>Due to the structural similarity between 5-mC and 5-hmC, these bases are experimentally almost indistinguishable. Recent articles demonstrated that the most common approaches (e.g. enzymatic approaches, bisulfite sequencing) do not account for 5-hmC. The development of the affinity-based technologies appears to be the most powerful way to differentially and specifically enrich 5-mC and 5-hmC sequences. The results shown here illustrate the use of this unique monoclonal antibody against 5-hydroxymethylcytosine that has been fully validated in various technologies.</p>',
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Preliminary results indicate that 5-hmC may have important roles distinct from 5-mC. Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics : 5-hydroxymethylcytosine may well represent a new pathway to demethylate DNA involving a repair mechanism converting 5-hmC to cytosine and, as such open up entirely new perspectives in epigenetic studies.
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<td>Fig 1</td>
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<tr>
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<td>Fig 2</td>
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<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig1.png" alt="hMeDIP" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<p><small><strong>Figure 1. Hydroxymethylated DNA IP results obtained with our hMeDIP kit (Cat. No. AF-104-0016)</strong><br /> Hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit. This kit includes: the monoclonal antibody against 5-hydroxymethylcytosine (Cat. No. MAb-633HMC-050), 5-hmC, 5-mC & cytosine DNA standards & Rat IgG (Cat. No. AF-105-0025). The DNA was prepared with the GenDNA module and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. 1 μg of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP’d material has been analysed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control • with Rat IgG as negative control (0.06%, almost no recovery) • with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control • with Rat IgG as negative control (0.03%, almost no recovery) • with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control • with Rat IgG as negative control (0.04%, almost no recovery) • with 5-hmC (97.60% recovery, almost full recovery) These results clearly demonstrate the high specificity and efficiency of the 5-hydroxymethylcytosine monoclonal antibody.</small></p>
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<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig2.png" alt="ELISA" style="display: block; margin-left: auto; margin-right: auto;" width="375" height="274" /></p>
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<div class="small-6 columns">
<p><small><strong>Figure 2. Determination of the 5-hmC rat monoclonal antibody titer</strong><br />To determine the titer, an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody directed against 5-hmC (Cat No. MAb-633HMC-050, MAb-633HMC-100) in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.</small></p>
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</div>
<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig3.png" alt="Dot blot" style="display: block; margin-left: auto; margin-right: auto;" width="190" height="192" /></p>
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<p><small><strong>Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls</strong><br />Figure 3A: Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.</small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig4.png" style="display: block; margin-left: auto; margin-right: auto;" width="115" height="232" /></p>
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<p><small><strong>Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls</strong><br />200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode « 5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.</small></p>
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'info2' => '<p>5-hydroxymethylcytosine (5-hmC) has been recently discovered in mammalian DNA. This results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. So far, the 5-hmC bases have been identified in Purkinje neurons, in granule cells and embryonic stem cells where they are present at high levels (up to 0,6% of total nucleotides in Purkinje cells).</p>
<p>Preliminary results indicate that 5-hmC may have important roles distinct from 5-mC. Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics : 5-hydroxymethylcytosine may well represent a new pathway to demethylate DNA involving a repair mechanism converting 5-hmC to cytosine and, as such open up entirely new perspectives in epigenetic studies.</p>
<p>Due to the structural similarity between 5-mC and 5-hmC, these bases are experimentally almost indistinguishable. Recent articles demonstrated that the most common approaches (e.g. enzymatic approaches, bisulfite sequencing) do not account for 5-hmC. The development of the affinity-based technologies appears to be the most powerful way to differentially and specifically enrich 5-mC and 5-hmC sequences. The results shown here illustrate the use of this unique monoclonal antibody against 5-hydroxymethylcytosine that has been fully validated in various technologies.</p>',
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<p style="text-align: center;"><strong>Make your Bisulfite conversion now in only 60 minutes !</strong></p>
<p>Diagenode's Premium Bisulfite Kit rapidly converts DNA through bisulfite treatment. Our conversion reagent is added directly to DNA, requires no intermediate steps, and results in high yields of DNA ready for downstream analysis methods including PCR and Next-Generation Sequencing.</p>',
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<p><em>Looking for hMeDIP-seq protocol? <a href="https://go.diagenode.com/l/928883/2022-01-07/2m1ht" target="_blank" title="Contact us">Contact us</a></em></p>
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'description' => '<p><span style="font-weight: 400;">T</span><span style="font-weight: 400;">he pattern of <strong>DNA modifications</strong> is critical for genome stability and the control of gene expression in the cell. Methylation of 5-cytosine (5-mC), one of the best-studied epigenetic marks, is carried out by the <strong>DNA methyltransferases</strong> DNMT3A and B and DNMT1. DNMT3A and DNMT3B are responsible for </span><i><span style="font-weight: 400;">de novo</span></i><span style="font-weight: 400;"> DNA methylation, whereas DNMT1 maintains existing methylation. 5-mC undergoes active demethylation which is performed by the <strong>Ten-Eleven Translocation</strong> (TET) familly of DNA hydroxylases. The latter consists of 3 members TET1, 2 and 3. All 3 members catalyze the conversion of <strong>5-methylcytosine</strong> (5-mC) into <strong>5-hydroxymethylcytosine</strong> (5-hmC), and further into <strong>5-formylcytosine</strong> (5-fC) and <strong>5-carboxycytosine</strong> (5-caC). 5-fC and 5-caC can be converted to unmodified cytosine by <strong>Thymine DNA Glycosylase</strong> (TDG). It is not yet clear if 5-hmC, 5-fC and 5-caC have specific functions or are simply intermediates in the demethylation of 5-mC.</span></p>
<p><span style="font-weight: 400;">DNA methylation is generally considered as a repressive mark and is usually associated with gene silencing. It is essential that the balance between DNA methylation and demethylation is precisely maintained. Dysregulation of DNA methylation may lead to many different human diseases and is often observed in cancer cells.</span></p>
<p><span style="font-weight: 400;">Diagenode offers highly validated antibodies against different proteins involved in DNA modifications as well as against the modified bases allowing the study of all steps and intermediates in the DNA methylation/demethylation pathway:</span></p>
<p><img src="https://www.diagenode.com/img/categories/antibodies/dna-methylation.jpg" height="599" width="816" /></p>
<p><strong>Diagenode exclusively sources the original 5-methylcytosine monoclonal antibody (clone 33D3).</strong></p>
<p>Check out the list below to see all proposed antibodies for DNA modifications.</p>
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<li>Highly sensitive and specific</li>
<li>Cost-effective (requires less antibody per reaction)</li>
<li>Batch-specific data is available on the website</li>
<li>Expert technical support</li>
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'name' => 'Exclusive Highly Specific Kits Antibodies for DNA HydroxyMethylation Studies',
'description' => '<p>Cytosine hydroxymethylation was recently discovered as an important epigenetic mechanism. This cytosine base modification results from the enzymatic conversion of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC) by the TET family of oxygenases. Though the precise role of 5-hmC is the subject of intense research and debate, early studies strongly indicate that it is also involved in gene regulation and in numerous important biological processes including embryonic development, cellular differentiation, stem cell reprogramming and carcinogenesis.</p>
<p>The study of 5-hmC has long been limited due to the lack of high quality, validated tools and technologies that discriminate hydroxymethylation from methylation in regulating gene expression. The use of highly specific antibodies against 5-hmC for the immunoprecipitation of hydroxymethylated DNA offers a reliable solution for hydroxymethylation profiling.</p>
<p></p>',
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'type' => 'Poster',
'url' => 'files/posters/Exclusive_Highly_Specific_Kits_Antibodies_for_DNA_HydroxyMethylation_Studies_Poster.pdf',
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'id' => '250',
'name' => 'product/antibodies/antibody.png',
'alt' => 'Mouse IgG',
'modified' => '2020-11-27 07:00:09',
'created' => '2015-07-17 10:12:18',
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'Protocol' => array(
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'id' => '15',
'name' => 'Dot blot protocol for 5-hydroxymethylcytosine monoclonal rat antibody',
'description' => '<div class="page" title="Page 1">
<div class="section">
<div class="layoutArea">
<div class="column">
<p><span> </span></p>
</div>
</div>
</div>
</div>',
'image_id' => null,
'type' => 'Protocol',
'url' => 'files/protocols/Protocol-Dot-blot5-hmc.pdf',
'slug' => 'protocol-dot-blot5-hmc',
'meta_keywords' => '',
'meta_description' => '',
'modified' => '2015-10-01 19:00:42',
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(int) 0 => array(
'id' => '4249',
'name' => 'DNMT1 regulates the timing of DNA methylation by DNMT3 in anenzymatic activity-dependent manner in mouse embryonic stem cells.',
'authors' => 'Ito Takamasa et al.',
'description' => '<p>DNA methylation (DNAme; 5-methylcytosine, 5mC) plays an essential role in mammalian development, and the 5mC profile is regulated by a balance of opposing enzymatic activities: DNA methyltransferases (DNMTs) and Ten-eleven translocation dioxygenases (TETs). In mouse embryonic stem cells (ESCs), de novo DNAme by DNMT3 family enzymes, demethylation by the TET-mediated conversion of 5mC to 5-hydroxymethylation (5hmC), and maintenance of the remaining DNAme by DNMT1 are actively repeated throughout cell cycles, dynamically forming a constant 5mC profile. Nevertheless, the detailed mechanism and physiological significance of this active cyclic DNA modification in mouse ESCs remain unclear. Here by visualizing the localization of DNA modifications on metaphase chromosomes and comparing whole-genome methylation profiles before and after the mid-S phase in ESCs lacking Dnmt1 (1KO ESCs), we demonstrated that in 1KO ESCs, DNMT3-mediated remethylation was interrupted during and after DNA replication. This results in a marked asymmetry in the distribution of 5hmC between sister chromatids at mitosis, with one chromatid being almost no 5hmC. When introduced in 1KO ESCs, the catalytically inactive form of DNMT1 (DNMT1CI) induced an increase in DNAme in pericentric heterochromatin and the DNAme-independent repression of IAPEz, a retrotransposon family, in 1KO ESCs. However, DNMT1CI could not restore the ability of DNMT3 to methylate unmodified dsDNA de novo in S phase in 1KO ESCs. Furthermore, during in vitro differentiation into epiblasts, 1KO ESCs expressing DNMT1CI showed an even stronger tendency to differentiate into the primitive endoderm than 1KO ESCs and were readily reprogrammed into the primitive streak via an epiblast-like cell state, reconfirming the importance of DNMT1 enzymatic activity at the onset of epiblast differentiation. These results indicate a novel function of DNMT1, in which DNMT1 actively regulates the timing and genomic targets of de novo methylation by DNMT3 in an enzymatic activity-dependent and independent manner, respectively.</p>',
'date' => '2022-01-01',
'pmid' => 'https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0262277',
'doi' => '10.1371/journal.pone.0262277',
'modified' => '2022-05-20 09:34:50',
'created' => '2022-05-19 10:41:50',
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(int) 1 => array(
'id' => '4045',
'name' => 'Functional role of Tet-mediated RNA hydroxymethylcytosine in mouse ES
cells and during differentiation.',
'authors' => 'Lan, Jie and Rajan, Nicholas and Bizet, Martin and Penning, Audrey and
Singh, Nitesh K and Guallar, Diana and Calonne, Emilie and Li Greci, Andrea
and Bonvin, Elise and Deplus, Rachel and Hsu, Phillip J and Nachtergaele,
Sigrid and Ma, Chengjie and Song, ',
'description' => 'Tet-enzyme-mediated 5-hydroxymethylation of cytosines in DNA plays a
crucial role in mouse embryonic stem cells (ESCs). In RNA also,
5-hydroxymethylcytosine (5hmC) has recently been evidenced, but its
physiological roles are still largely unknown. Here we show the
contribution and function of this mark in mouse ESCs and differentiating
embryoid bodies. Transcriptome-wide mapping in ESCs reveals hundreds of
messenger RNAs marked by 5hmC at sites characterized by a defined unique
consensus sequence and particular features. During differentiation a large
number of transcripts, including many encoding key pluripotency-related
factors (such as Eed and Jarid2), show decreased cytosine
hydroxymethylation. Using Tet-knockout ESCs, we find Tet enzymes to be
partly responsible for deposition of 5hmC in mRNA. A transcriptome-wide
search further reveals mRNA targets to which Tet1 and Tet2 bind, at sites
showing a topology similar to that of 5hmC sites. Tet-mediated RNA
hydroxymethylation is found to reduce the stability of crucial
pluripotency-promoting transcripts. We propose that RNA cytosine
5-hydroxymethylation by Tets is a mark of transcriptome flexibility,
inextricably linked to the balance between pluripotency and lineage
commitment.',
'date' => '2020-10-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/33009383',
'doi' => '10.1038/s41467-020-18729-6',
'modified' => '2021-02-18 10:21:53',
'created' => '2021-02-18 10:21:53',
'ProductsPublication' => array(
[maximum depth reached]
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),
(int) 2 => array(
'id' => '3660',
'name' => 'Global distribution of DNA hydroxymethylation and DNA methylation in chronic lymphocytic leukemia.',
'authors' => 'Wernig-Zorc S, Yadav MP, Kopparapu PK, Bemark M, Kristjansdottir HL, Andersson PO, Kanduri C, Kanduri M',
'description' => '<p>BACKGROUND: Chronic lymphocytic leukemia (CLL) has been a good model system to understand the functional role of 5-methylcytosine (5-mC) in cancer progression. More recently, an oxidized form of 5-mC, 5-hydroxymethylcytosine (5-hmC) has gained lot of attention as a regulatory epigenetic modification with prognostic and diagnostic implications for several cancers. However, there is no global study exploring the role of 5-hydroxymethylcytosine (5-hmC) levels in CLL. Herein, using mass spectrometry and hMeDIP-sequencing, we analysed the dynamics of 5-hmC during B cell maturation and CLL pathogenesis. RESULTS: We show that naïve B-cells had higher levels of 5-hmC and 5-mC compared to non-class switched and class-switched memory B-cells. We found a significant decrease in global 5-mC levels in CLL patients (n = 15) compared to naïve and memory B cells, with no changes detected between the CLL prognostic groups. On the other hand, global 5-hmC levels of CLL patients were similar to memory B cells and reduced compared to naïve B cells. Interestingly, 5-hmC levels were increased at regulatory regions such as gene-body, CpG island shores and shelves and 5-hmC distribution over the gene-body positively correlated with degree of transcriptional activity. Importantly, CLL samples showed aberrant 5-hmC and 5-mC pattern over gene-body compared to well-defined patterns in normal B-cells. Integrated analysis of 5-hmC and RNA-sequencing from CLL datasets identified three novel oncogenic drivers that could have potential roles in CLL development and progression. CONCLUSIONS: Thus, our study suggests that the global loss of 5-hmC, accompanied by its significant increase at the gene regulatory regions, constitute a novel hallmark of CLL pathogenesis. Our combined analysis of 5-mC and 5-hmC sequencing provided insights into the potential role of 5-hmC in modulating gene expression changes during CLL pathogenesis.</p>',
'date' => '2019-01-07',
'pmid' => 'http://www.pubmed.gov/30616658',
'doi' => '10.1186/s13072‑018‑0252‑7',
'modified' => '2019-07-01 11:46:16',
'created' => '2019-06-21 14:55:31',
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),
(int) 3 => array(
'id' => '2992',
'name' => 'Regulation of the DNA Methylation Landscape in Human Somatic Cell Reprogramming by the miR-29 Family',
'authors' => 'Hysolli E et al.',
'description' => 'Reprogramming to pluripotency after overexpression of OCT4, SOX2, KLF4, and MYC is accompanied by global genomic and epigenomic changes. Histone modification and DNA methylation states in induced pluripotent stem cells (iPSCs) have been shown to be highly similar to embryonic stem cells (ESCs). However, epigenetic differences still exist between iPSCs and ESCs. In particular, aberrant DNA methylation states found in iPSCs are a major concern when using iPSCs in a clinical setting. Thus, it is critical to find factors that regulate DNA methylation states in reprogramming. Here, we found that the miR-29 family is an important epigenetic regulator during human somatic cell reprogramming. Our global DNA methylation and hydroxymethylation analysis shows that DNA demethylation is a major event mediated by miR-29a depletion during early reprogramming, and that iPSCs derived from miR-29a depletion are epigenetically closer to ESCs. Our findings uncover an important miRNA-based approach to generate clinically robust iPSCs.',
'date' => '2016-07-12',
'pmid' => 'http://www.ncbi.nlm.nih.gov/pubmed/27373925',
'doi' => '10.1016/j.stemcr.2016.05.014',
'modified' => '2016-08-23 09:57:29',
'created' => '2016-08-23 09:57:29',
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(int) 4 => array(
'id' => '2811',
'name' => 'Transcriptome-wide distribution and function of RNA hydroxymethylcytosine',
'authors' => 'Delatte B, Wang F, Ngoc LV, Collignon E, Bonvin E, Deplus R, Calonne E, Hassabi B, Putmans P, Awe S, Wetzel C, Kreher J, Soin R, Creppe C, Limbach PA, Gueydan C, Kruys V, Brehm A, Minakhina S, Defrance M, Steward R, Fuks F.',
'description' => '<p>Hydroxymethylcytosine, well described in DNA, occurs also in RNA. Here, we show that hydroxymethylcytosine preferentially marks polyadenylated RNAs and is deposited by Tet in Drosophila. We map the transcriptome-wide hydroxymethylation landscape, revealing hydroxymethylcytosine in the transcripts of many genes, notably in coding sequences, and identify consensus sites for hydroxymethylation. We found that RNA hydroxymethylation can favor mRNA translation. Tet and hydroxymethylated RNA are found to be most abundant in the Drosophila brain, and Tet-deficient fruitflies suffer impaired brain development, accompanied by decreased RNA hydroxymethylation. This study highlights the distribution, localization, and function of cytosine hydroxymethylation and identifies central roles for this modification in Drosophila.</p>',
'date' => '2016-01-15',
'pmid' => 'http://pubmed.gov/26816380',
'doi' => '10.1126/science.aac5253',
'modified' => '2016-01-28 21:57:55',
'created' => '2016-01-28 21:57:55',
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[maximum depth reached]
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),
(int) 5 => array(
'id' => '3750',
'name' => 'RNA biochemistry. Transcriptome-wide distribution and function of RNA hydroxymethylcytosine.',
'authors' => 'Delatte B, Wang F, Ngoc LV, Collignon E, Bonvin E, Deplus R, Calonne E, Hassabi B, Putmans P, Awe S, Wetzel C, Kreher J, Soin R, Creppe C, Limbach PA, Gueydan C, Kruys V, Brehm A, Minakhina S, Defrance M, Steward R, Fuks F',
'description' => '<p>Hydroxymethylcytosine, well described in DNA, occurs also in RNA. Here, we show that hydroxymethylcytosine preferentially marks polyadenylated RNAs and is deposited by Tet in Drosophila. We map the transcriptome-wide hydroxymethylation landscape, revealing hydroxymethylcytosine in the transcripts of many genes, notably in coding sequences, and identify consensus sites for hydroxymethylation. We found that RNA hydroxymethylation can favor mRNA translation. Tet and hydroxymethylated RNA are found to be most abundant in the Drosophila brain, and Tet-deficient fruitflies suffer impaired brain development, accompanied by decreased RNA hydroxymethylation. This study highlights the distribution, localization, and function of cytosine hydroxymethylation and identifies central roles for this modification in Drosophila.</p>',
'date' => '2016-01-15',
'pmid' => 'http://www.pubmed.org/26816380',
'doi' => '10.1126/science.aac5253',
'modified' => '2019-10-03 12:29:05',
'created' => '2019-10-02 16:16:55',
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[maximum depth reached]
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),
(int) 6 => array(
'id' => '2613',
'name' => 'CpG signalling, H2A.Z/H3 acetylation and microRNA-mediated deferred self-attenuation orchestrate foetal NOS3 expression.',
'authors' => 'Postberg J, Kanders M, Forcob S, Willems R, Orth V, Hensel KO, Weil PP, Wirth S, Jenke AC',
'description' => 'BACKGROUND: An adverse intrauterine environment leads to permanent physiological changes including vascular tone regulation, potentially influencing the risk for adult vascular diseases. We therefore aimed to monitor responsive NOS3 expression in human umbilical artery endothelial cells (HUAEC) and to study the underlying epigenetic signatures involved in its regulation. RESULTS: NOS3 and STAT3 mRNA levels were elevated in HUAEC of patients who suffered from placental insufficiency. 5-hydroxymethylcytosine, H3K9ac and Histone 2A (H2A).Zac at the NOS3 transcription start site directly correlated with NOS3 mRNA levels. Concomitantly, we observed entangled histone acetylation patterns and NOS3 response upon hypoxic conditions in vitro. Knock-down of either NOS3 or STAT3 by RNAi provided evidence for a functional NOS3/STAT3 relationship. Moreover, we recognized massive turnover of Stat3 at a discrete binding site in the NOS3 promoter. Interestingly, induced hyperacetylation resulted in short-termed increase of NOS3 mRNA followed by deferred decrease indicating that NOS3 expression could become self-attenuated by co-expressed intronic 27 nt-ncRNA. Reporter assay results and phylogenetic analyses enabled us to propose a novel model for STAT3-3'-UTR targeting by this 27-nt-ncRNA. CONCLUSIONS: An adverse intrauterine environment leads to adaptive changes of NOS3 expression. Apparently, a rapid NOS3 self-limiting response upon ectopic triggers co-exists with longer termed expression changes in response to placental insufficiency involving differential epigenetic signatures. Their persistence might contribute to impaired vascular endothelial response and consequently increase the risk of cardiovascular disease later in life.',
'date' => '2015-01-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/25699114',
'doi' => '',
'modified' => '2015-07-24 15:39:05',
'created' => '2015-07-24 15:39:05',
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[maximum depth reached]
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),
(int) 7 => array(
'id' => '2348',
'name' => 'White matter tract and glial-associated changes in 5-hydroxymethylcytosine following chronic cerebral hypoperfusion.',
'authors' => 'Tsenkina Y, Ruzov A, Gliddon C, Horsburgh K, De Sousa PA',
'description' => 'White matter abnormalities due to age-related cerebrovascular alterations is a common pathological hallmark associated with functional impairment in the elderly which has been modeled in chronically hypoperfused mice. 5-Methylcytosine (5mC) and its oxidized derivative 5-hydroxymethylcytosine (5hmC) are DNA modifications that have been recently linked with age-related neurodegeneration and cerebrovascular pathology. Here we conducted a pilot investigation of whether chronic cerebral hypoperfusion might affect genomic distribution of these modifications and/ or a Ten-Eleven Translocation protein 2 (TET2) which catalyses hydroxymethylation in white and grey matter regions of this animal model. Immunohistochemical evaluation of sham and chronically hypoperfused mice a month after surgery revealed significant (p<0.05) increases in the proportion of 5hmC positive cells, Iba1 positive inflammatory microglia, and NG2 positive oligodendroglial progenitors in the hypoperfused corpus callosum. In the same white matter tract there was an absence of hypoperfusion-induced alterations in the proportion of 5mC, TET2 positive cells and CC1 positive mature oligodrendrocytes. Correlation analysis across animals within both treatment groups demonstrated a significant association of the elevated 5hmC levels with increases in the proportion of inflammatory microglia only (p=0.01) in the corpus callosum. In vitro studies revealed that 5hmC is lost during oligodendroglial maturation but not microglial activation. Additionally, TET1, TET2, and TET3 protein levels showed dynamic alterations during oligodendroglial development and following oxidative stress in vitro. Our study suggests that 5hmC exhibits white matter tract and cell type specific dynamics following chronic cerebral hypoperfusion in mice.',
'date' => '2014-10-10',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/25305569',
'doi' => '',
'modified' => '2015-07-24 15:39:04',
'created' => '2015-07-24 15:39:04',
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(int) 8 => array(
'id' => '994',
'name' => 'Tet2 Facilitates the Derepression of Myeloid Target Genes during CEBPα-Induced Transdifferentiation of Pre-B Cells.',
'authors' => 'Kallin EM, Rodríguez-Ubreva J, Christensen J, Cimmino L, Aifantis I, Helin K, Ballestar E, Graf T',
'description' => 'The methylcytosine hydroxylase Tet2 has been implicated in hematopoietic differentiation and the formation of myeloid malignancies when mutated. An ideal system to study the role of Tet2 in myelopoeisis is CEBPα-induced transdifferentiation of pre-B cells into macrophages. Here we found that CEBPα binds to upstream regions of Tet2 and that the gene becomes activated. Tet2 knockdowns impaired the upregulation of macrophage markers as well as phagocytic capacity, suggesting that the enzyme is required for both early and late stage myeloid differentiation. A slightly weaker effect was seen in primary cells with a Tet2 ablation. Expression arrays of transdifferentiating cells with Tet2 knockdowns permitted the identification of a small subset of myeloid genes whose upregulation was blunted. Activation of these target genes was accompanied by rapid increases of promoter hydroxy-methylation. Our observations indicate that Tet2 helps CEBPα rapidly derepress myeloid genes during the conversion of pre-B cells into macrophages.',
'date' => '2012-09-12',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/22981865',
'doi' => '',
'modified' => '2015-07-24 15:38:59',
'created' => '2015-07-24 15:38:59',
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[maximum depth reached]
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(int) 9 => array(
'id' => '149',
'name' => 'Lineage-specific distribution of high levels of genomic 5-hydroxymethylcytosine in mammalian development.',
'authors' => 'Ruzov A, Tsenkina Y, Serio A, Dudnakova T, Fletcher J, Bai Y, Chebotareva T, Pells S, Hannoun Z, Sullivan G, Chandran S, Hay DC, Bradley M, Wilmut I, De Sousa P',
'description' => 'Methylation of cytosine is a DNA modification associated with gene repression. Recently, a novel cytosine modification, 5-hydroxymethylcytosine (5-hmC) has been discovered. Here we examine 5-hmC distribution during mammalian development and in cellular systems, and show that the developmental dynamics of 5-hmC are different from those of 5-methylcytosine (5-mC); in particular 5-hmC is enriched in embryonic contexts compared to adult tissues. A detectable 5-hmC signal appears in pre-implantation development starting at the zygote stage, where the paternal genome is subjected to a genome-wide hydroxylation of 5-mC, which precisely coincides with the loss of the 5-mC signal in the paternal pronucleus. Levels of 5-hmC are high in cells of the inner cell mass in blastocysts, and the modification colocalises with nestin-expressing cell populations in mouse post-implantation embryos. Compared to other adult mammalian organs, 5-hmC is strongly enriched in bone marrow and brain, wherein high 5-hmC content is a feature of both neuronal progenitors and post-mitotic neurons. We show that high levels of 5-hmC are not only present in mouse and human embryonic stem cells (ESCs) and lost during differentiation, as has been reported previously, but also reappear during the generation of induced pluripotent stem cells; thus 5-hmC enrichment correlates with a pluripotent cell state. Our findings suggest that apart from the cells of neuronal lineages, high levels of genomic 5-hmC are an epigenetic feature of embryonic cell populations and cellular pluri- and multi-lineage potency. To our knowledge, 5-hmC represents the first epigenetic modification of DNA discovered whose enrichment is so cell-type specific.',
'date' => '2011-09-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/21747414',
'doi' => '',
'modified' => '2015-07-24 15:38:57',
'created' => '2015-07-24 15:38:57',
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[maximum depth reached]
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(int) 10 => array(
'id' => '361',
'name' => 'Genome-wide analysis of 5-hydroxymethylcytosine distribution reveals its dual function in transcriptional regulation in mouse embryonic stem cells.',
'authors' => 'Wu H, D'Alessio AC, Ito S, Wang Z, Cui K, Zhao K, Sun YE, Zhang Y',
'description' => 'Recent studies have demonstrated that the Ten-eleven translocation (Tet) family proteins can enzymatically convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). While 5mC has been studied extensively, little is known about the distribution and function of 5hmC. Here we present a genome-wide profile of 5hmC in mouse embryonic stem (ES) cells. A combined analysis of global 5hmC distribution and gene expression profile in wild-type and Tet1-depleted ES cells suggests that 5hmC is enriched at both gene bodies of actively transcribed genes and extended promoter regions of Polycomb-repressed developmental regulators. Thus, our study reveals the first genome-wide 5hmC distribution in pluripotent stem cells, and supports its dual function in regulating gene expression.',
'date' => '2011-04-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/21460036',
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'name' => '5-hydroxymethylcytosine (5-hmC) Antibody (rat) ',
'description' => '<p>5<span>-hmC is a DNA modification which results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. Preliminary results indicate that 5-hmC may have important roles distinct from 5-methylcytosine (5-mC). Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics.</span></p>',
'label1' => 'Validation Data',
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<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig1.png" alt="hMeDIP" style="display: block; margin-left: auto; margin-right: auto;" /></p>
</div>
<div class="small-6 columns">
<p><small><strong>Figure 1. Hydroxymethylated DNA IP results obtained with our hMeDIP kit (Cat. No. AF-104-0016)</strong><br /> Hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit. This kit includes: the monoclonal antibody against 5-hydroxymethylcytosine (Cat. No. MAb-633HMC-050), 5-hmC, 5-mC & cytosine DNA standards & Rat IgG (Cat. No. AF-105-0025). The DNA was prepared with the GenDNA module and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. 1 μg of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP’d material has been analysed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control • with Rat IgG as negative control (0.06%, almost no recovery) • with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control • with Rat IgG as negative control (0.03%, almost no recovery) • with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control • with Rat IgG as negative control (0.04%, almost no recovery) • with 5-hmC (97.60% recovery, almost full recovery) These results clearly demonstrate the high specificity and efficiency of the 5-hydroxymethylcytosine monoclonal antibody.</small></p>
</div>
</div>
<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig2.png" alt="ELISA" style="display: block; margin-left: auto; margin-right: auto;" width="375" height="274" /></p>
</div>
<div class="small-6 columns">
<p><small><strong>Figure 2. Determination of the 5-hmC rat monoclonal antibody titer</strong><br />To determine the titer, an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody directed against 5-hmC (Cat No. MAb-633HMC-050, MAb-633HMC-100) in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.</small></p>
</div>
</div>
<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig3.png" alt="Dot blot" style="display: block; margin-left: auto; margin-right: auto;" width="190" height="192" /></p>
</div>
<div class="small-6 columns">
<p><small><strong>Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls</strong><br />Figure 3A: Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.</small></p>
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<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig4.png" style="display: block; margin-left: auto; margin-right: auto;" width="115" height="232" /></p>
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<p><small><strong>Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls</strong><br />200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode « 5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.</small></p>
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'info2' => '<p>5-hydroxymethylcytosine (5-hmC) has been recently discovered in mammalian DNA. This results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. So far, the 5-hmC bases have been identified in Purkinje neurons, in granule cells and embryonic stem cells where they are present at high levels (up to 0,6% of total nucleotides in Purkinje cells).</p>
<p>Preliminary results indicate that 5-hmC may have important roles distinct from 5-mC. Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics : 5-hydroxymethylcytosine may well represent a new pathway to demethylate DNA involving a repair mechanism converting 5-hmC to cytosine and, as such open up entirely new perspectives in epigenetic studies.</p>
<p>Due to the structural similarity between 5-mC and 5-hmC, these bases are experimentally almost indistinguishable. Recent articles demonstrated that the most common approaches (e.g. enzymatic approaches, bisulfite sequencing) do not account for 5-hmC. The development of the affinity-based technologies appears to be the most powerful way to differentially and specifically enrich 5-mC and 5-hmC sequences. The results shown here illustrate the use of this unique monoclonal antibody against 5-hydroxymethylcytosine that has been fully validated in various technologies.</p>',
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'name' => '5-hydroxymethylcytosine (5-hmC) Antibody (rat) (sample size)',
'description' => '<p><span>5-hmC is a DNA modification which results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. Preliminary results indicate that 5-hmC may have important roles distinct from 5-methylcytosine (5-mC). Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics.</span></p>',
'label1' => 'Validation Data',
'info1' => '<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig1.png" alt="hMeDIP" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 1. Hydroxymethylated DNA IP results obtained with our hMeDIP kit (Cat. No. AF-104-0016)</strong><br /> Hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit. This kit includes: the monoclonal antibody against 5-hydroxymethylcytosine (Cat. No. MAb-633HMC-050), 5-hmC, 5-mC & cytosine DNA standards & Rat IgG (Cat. No. AF-105-0025). The DNA was prepared with the GenDNA module and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. 1 μg of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP’d material has been analysed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control • with Rat IgG as negative control (0.06%, almost no recovery) • with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control • with Rat IgG as negative control (0.03%, almost no recovery) • with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control • with Rat IgG as negative control (0.04%, almost no recovery) • with 5-hmC (97.60% recovery, almost full recovery) These results clearly demonstrate the high specificity and efficiency of the 5-hydroxymethylcytosine monoclonal antibody.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig2.png" alt="ELISA" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 2. Determination of the 5-hmC rat monoclonal antibody titer</strong><br /> To determine the titer, an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody directed against 5-hmC (Cat No. MAb-633HMC-050, MAb-633HMC-100) in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig3.png" alt="Dot blot" style="display: block; margin-left: auto; margin-right: auto;" width="200" height="200" /></p>
<p><strong>Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls</strong><br /> Figure 3A: Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig4.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls</strong><br /> 200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode « 5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.</p>',
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'name' => '5-hydroxymethylcytosine (5-hmC) Antibody (rat) ',
'description' => '<p>5<span>-hmC is a DNA modification which results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. Preliminary results indicate that 5-hmC may have important roles distinct from 5-methylcytosine (5-mC). Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics.</span></p>',
'label1' => 'Validation Data',
'info1' => '<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig1.png" alt="hMeDIP" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 1. Hydroxymethylated DNA IP results obtained with our hMeDIP kit (Cat. No. AF-104-0016)</strong><br /> Hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit. This kit includes: the monoclonal antibody against 5-hydroxymethylcytosine (Cat. No. MAb-633HMC-050), 5-hmC, 5-mC & cytosine DNA standards & Rat IgG (Cat. No. AF-105-0025). The DNA was prepared with the GenDNA module and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. 1 μg of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP’d material has been analysed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control • with Rat IgG as negative control (0.06%, almost no recovery) • with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control • with Rat IgG as negative control (0.03%, almost no recovery) • with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control • with Rat IgG as negative control (0.04%, almost no recovery) • with 5-hmC (97.60% recovery, almost full recovery) These results clearly demonstrate the high specificity and efficiency of the 5-hydroxymethylcytosine monoclonal antibody.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig2.png" alt="ELISA" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 2. Determination of the 5-hmC rat monoclonal antibody titer</strong><br /> To determine the titer, an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody directed against 5-hmC (Cat No. MAb-633HMC-050, MAb-633HMC-100) in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig3.png" alt="Dot blot" style="display: block; margin-left: auto; margin-right: auto;" width="200" height="200" /></p>
<p><strong>Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls</strong><br /> Figure 3A: Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig4.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls</strong><br /> 200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode « 5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.</p>',
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<p><span>The hMeDIP kit is designed for enrichment of hydroxymethylated DNA from fragmented genomic DNA<span><span> </span>samples for use in genome-wide methylation analysis. It features</span></span><span> a highly specific monoclonal antibody against </span>5-hydroxymethylcytosine (5-hmC) for the immunoprecipitation of hydroxymethylated DNA<span>. It includes control DNA and primers to assess the effiency of the assay. </span>Performing hydroxymethylation profiling with the hMeDIP kit is fast, reliable and highly specific.</p>
<p><em>Looking for hMeDIP-seq protocol? <a href="https://go.diagenode.com/l/928883/2022-01-07/2m1ht" target="_blank" title="Contact us">Contact us</a></em></p>
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'description' => '<p><span>5-hmC is a DNA modification which results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. Preliminary results indicate that 5-hmC may have important roles distinct from 5-methylcytosine (5-mC). Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics.</span></p>',
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'info1' => '<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig1.png" alt="hMeDIP" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 1. Hydroxymethylated DNA IP results obtained with our hMeDIP kit (Cat. No. AF-104-0016)</strong><br /> Hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit. This kit includes: the monoclonal antibody against 5-hydroxymethylcytosine (Cat. No. MAb-633HMC-050), 5-hmC, 5-mC & cytosine DNA standards & Rat IgG (Cat. No. AF-105-0025). The DNA was prepared with the GenDNA module and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. 1 μg of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP’d material has been analysed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control • with Rat IgG as negative control (0.06%, almost no recovery) • with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control • with Rat IgG as negative control (0.03%, almost no recovery) • with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control • with Rat IgG as negative control (0.04%, almost no recovery) • with 5-hmC (97.60% recovery, almost full recovery) These results clearly demonstrate the high specificity and efficiency of the 5-hydroxymethylcytosine monoclonal antibody.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig2.png" alt="ELISA" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 2. Determination of the 5-hmC rat monoclonal antibody titer</strong><br /> To determine the titer, an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody directed against 5-hmC (Cat No. MAb-633HMC-050, MAb-633HMC-100) in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig3.png" alt="Dot blot" style="display: block; margin-left: auto; margin-right: auto;" width="200" height="200" /></p>
<p><strong>Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls</strong><br /> Figure 3A: Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig4.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls</strong><br /> 200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode « 5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.</p>',
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'name' => 'Exclusive Highly Specific Kits Antibodies for DNA HydroxyMethylation Studies',
'description' => '<p>Cytosine hydroxymethylation was recently discovered as an important epigenetic mechanism. This cytosine base modification results from the enzymatic conversion of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC) by the TET family of oxygenases. Though the precise role of 5-hmC is the subject of intense research and debate, early studies strongly indicate that it is also involved in gene regulation and in numerous important biological processes including embryonic development, cellular differentiation, stem cell reprogramming and carcinogenesis.</p>
<p>The study of 5-hmC has long been limited due to the lack of high quality, validated tools and technologies that discriminate hydroxymethylation from methylation in regulating gene expression. The use of highly specific antibodies against 5-hmC for the immunoprecipitation of hydroxymethylated DNA offers a reliable solution for hydroxymethylation profiling.</p>
<p></p>',
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'authors' => 'Wu H, D'Alessio AC, Ito S, Wang Z, Cui K, Zhao K, Sun YE, Zhang Y',
'description' => 'Recent studies have demonstrated that the Ten-eleven translocation (Tet) family proteins can enzymatically convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). While 5mC has been studied extensively, little is known about the distribution and function of 5hmC. Here we present a genome-wide profile of 5hmC in mouse embryonic stem (ES) cells. A combined analysis of global 5hmC distribution and gene expression profile in wild-type and Tet1-depleted ES cells suggests that 5hmC is enriched at both gene bodies of actively transcribed genes and extended promoter regions of Polycomb-repressed developmental regulators. Thus, our study reveals the first genome-wide 5hmC distribution in pluripotent stem cells, and supports its dual function in regulating gene expression.',
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'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/21460036',
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'description' => '<p>5<span>-hmC is a DNA modification which results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. Preliminary results indicate that 5-hmC may have important roles distinct from 5-methylcytosine (5-mC). Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics.</span></p>',
'label1' => 'Validation Data',
'info1' => '<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig1.png" alt="hMeDIP" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 1. Hydroxymethylated DNA IP results obtained with our hMeDIP kit (Cat. No. AF-104-0016)</strong><br /> Hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit. This kit includes: the monoclonal antibody against 5-hydroxymethylcytosine (Cat. No. MAb-633HMC-050), 5-hmC, 5-mC & cytosine DNA standards & Rat IgG (Cat. No. AF-105-0025). The DNA was prepared with the GenDNA module and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. 1 μg of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP’d material has been analysed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control • with Rat IgG as negative control (0.06%, almost no recovery) • with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control • with Rat IgG as negative control (0.03%, almost no recovery) • with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control • with Rat IgG as negative control (0.04%, almost no recovery) • with 5-hmC (97.60% recovery, almost full recovery) These results clearly demonstrate the high specificity and efficiency of the 5-hydroxymethylcytosine monoclonal antibody.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig2.png" alt="ELISA" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 2. Determination of the 5-hmC rat monoclonal antibody titer</strong><br /> To determine the titer, an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody directed against 5-hmC (Cat No. MAb-633HMC-050, MAb-633HMC-100) in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig3.png" alt="Dot blot" style="display: block; margin-left: auto; margin-right: auto;" width="200" height="200" /></p>
<p><strong>Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls</strong><br /> Figure 3A: Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig4.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls</strong><br /> 200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode « 5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.</p>',
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'description' => '<p>5<span>-hmC is a DNA modification which results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. Preliminary results indicate that 5-hmC may have important roles distinct from 5-methylcytosine (5-mC). Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics.</span></p>',
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<p><strong>Figure 1. Hydroxymethylated DNA IP results obtained with our hMeDIP kit (Cat. No. AF-104-0016)</strong><br /> Hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit. This kit includes: the monoclonal antibody against 5-hydroxymethylcytosine (Cat. No. MAb-633HMC-050), 5-hmC, 5-mC & cytosine DNA standards & Rat IgG (Cat. No. AF-105-0025). The DNA was prepared with the GenDNA module and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. 1 μg of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP’d material has been analysed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control • with Rat IgG as negative control (0.06%, almost no recovery) • with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control • with Rat IgG as negative control (0.03%, almost no recovery) • with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control • with Rat IgG as negative control (0.04%, almost no recovery) • with 5-hmC (97.60% recovery, almost full recovery) These results clearly demonstrate the high specificity and efficiency of the 5-hydroxymethylcytosine monoclonal antibody.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig2.png" alt="ELISA" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 2. Determination of the 5-hmC rat monoclonal antibody titer</strong><br /> To determine the titer, an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody directed against 5-hmC (Cat No. MAb-633HMC-050, MAb-633HMC-100) in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.</p>
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<p><strong>Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls</strong><br /> Figure 3A: Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig4.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls</strong><br /> 200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode « 5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.</p>',
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<p>Preliminary results indicate that 5-hmC may have important roles distinct from 5-mC. Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics : 5-hydroxymethylcytosine may well represent a new pathway to demethylate DNA involving a repair mechanism converting 5-hmC to cytosine and, as such open up entirely new perspectives in epigenetic studies.</p>
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Preliminary results indicate that 5-hmC may have important roles distinct from 5-mC. Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics : 5-hydroxymethylcytosine may well represent a new pathway to demethylate DNA involving a repair mechanism converting 5-hmC to cytosine and, as such open up entirely new perspectives in epigenetic studies.
Due to the structural similarity between 5-mC and 5-hmC, these bases are experimentally almost indistinguishable. Recent articles demonstrated that the most common approaches (e.g. enzymatic approaches, bisulfite sequencing) do not account for 5-hmC. The development of the affinity-based technologies appears to be the most powerful way to differentially and specifically enrich 5-mC and 5-hmC sequences. The results shown here illustrate the use of this unique monoclonal antibody against 5-hydroxymethylcytosine that has been fully validated in various technologies.',
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'description' => '<p>5<span>-hmC is a DNA modification which results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. Preliminary results indicate that 5-hmC may have important roles distinct from 5-methylcytosine (5-mC). Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics.</span></p>',
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig1.png" alt="hMeDIP" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<p><small><strong>Figure 1. Hydroxymethylated DNA IP results obtained with our hMeDIP kit (Cat. No. AF-104-0016)</strong><br /> Hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit. This kit includes: the monoclonal antibody against 5-hydroxymethylcytosine (Cat. No. MAb-633HMC-050), 5-hmC, 5-mC & cytosine DNA standards & Rat IgG (Cat. No. AF-105-0025). The DNA was prepared with the GenDNA module and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. 1 μg of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP’d material has been analysed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control • with Rat IgG as negative control (0.06%, almost no recovery) • with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control • with Rat IgG as negative control (0.03%, almost no recovery) • with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control • with Rat IgG as negative control (0.04%, almost no recovery) • with 5-hmC (97.60% recovery, almost full recovery) These results clearly demonstrate the high specificity and efficiency of the 5-hydroxymethylcytosine monoclonal antibody.</small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig2.png" alt="ELISA" style="display: block; margin-left: auto; margin-right: auto;" width="375" height="274" /></p>
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<p><small><strong>Figure 2. Determination of the 5-hmC rat monoclonal antibody titer</strong><br />To determine the titer, an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody directed against 5-hmC (Cat No. MAb-633HMC-050, MAb-633HMC-100) in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.</small></p>
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<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig3.png" alt="Dot blot" style="display: block; margin-left: auto; margin-right: auto;" width="190" height="192" /></p>
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<p><small><strong>Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls</strong><br />Figure 3A: Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.</small></p>
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<p><small><strong>Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls</strong><br />200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode « 5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.</small></p>
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<p>Preliminary results indicate that 5-hmC may have important roles distinct from 5-mC. Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics : 5-hydroxymethylcytosine may well represent a new pathway to demethylate DNA involving a repair mechanism converting 5-hmC to cytosine and, as such open up entirely new perspectives in epigenetic studies.</p>
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<p style="text-align: center;"><strong>Make your Bisulfite conversion now in only 60 minutes !</strong></p>
<p>Diagenode's Premium Bisulfite Kit rapidly converts DNA through bisulfite treatment. Our conversion reagent is added directly to DNA, requires no intermediate steps, and results in high yields of DNA ready for downstream analysis methods including PCR and Next-Generation Sequencing.</p>',
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<p><em>Looking for hMeDIP-seq protocol? <a href="https://go.diagenode.com/l/928883/2022-01-07/2m1ht" target="_blank" title="Contact us">Contact us</a></em></p>
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'description' => '<p><span style="font-weight: 400;">T</span><span style="font-weight: 400;">he pattern of <strong>DNA modifications</strong> is critical for genome stability and the control of gene expression in the cell. Methylation of 5-cytosine (5-mC), one of the best-studied epigenetic marks, is carried out by the <strong>DNA methyltransferases</strong> DNMT3A and B and DNMT1. DNMT3A and DNMT3B are responsible for </span><i><span style="font-weight: 400;">de novo</span></i><span style="font-weight: 400;"> DNA methylation, whereas DNMT1 maintains existing methylation. 5-mC undergoes active demethylation which is performed by the <strong>Ten-Eleven Translocation</strong> (TET) familly of DNA hydroxylases. The latter consists of 3 members TET1, 2 and 3. All 3 members catalyze the conversion of <strong>5-methylcytosine</strong> (5-mC) into <strong>5-hydroxymethylcytosine</strong> (5-hmC), and further into <strong>5-formylcytosine</strong> (5-fC) and <strong>5-carboxycytosine</strong> (5-caC). 5-fC and 5-caC can be converted to unmodified cytosine by <strong>Thymine DNA Glycosylase</strong> (TDG). It is not yet clear if 5-hmC, 5-fC and 5-caC have specific functions or are simply intermediates in the demethylation of 5-mC.</span></p>
<p><span style="font-weight: 400;">DNA methylation is generally considered as a repressive mark and is usually associated with gene silencing. It is essential that the balance between DNA methylation and demethylation is precisely maintained. Dysregulation of DNA methylation may lead to many different human diseases and is often observed in cancer cells.</span></p>
<p><span style="font-weight: 400;">Diagenode offers highly validated antibodies against different proteins involved in DNA modifications as well as against the modified bases allowing the study of all steps and intermediates in the DNA methylation/demethylation pathway:</span></p>
<p><img src="https://www.diagenode.com/img/categories/antibodies/dna-methylation.jpg" height="599" width="816" /></p>
<p><strong>Diagenode exclusively sources the original 5-methylcytosine monoclonal antibody (clone 33D3).</strong></p>
<p>Check out the list below to see all proposed antibodies for DNA modifications.</p>
<p>Diagenode’s highly validated antibodies:</p>
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<li>Cost-effective (requires less antibody per reaction)</li>
<li>Batch-specific data is available on the website</li>
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<p>The study of 5-hmC has long been limited due to the lack of high quality, validated tools and technologies that discriminate hydroxymethylation from methylation in regulating gene expression. The use of highly specific antibodies against 5-hmC for the immunoprecipitation of hydroxymethylated DNA offers a reliable solution for hydroxymethylation profiling.</p>
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'name' => 'DNMT1 regulates the timing of DNA methylation by DNMT3 in anenzymatic activity-dependent manner in mouse embryonic stem cells.',
'authors' => 'Ito Takamasa et al.',
'description' => '<p>DNA methylation (DNAme; 5-methylcytosine, 5mC) plays an essential role in mammalian development, and the 5mC profile is regulated by a balance of opposing enzymatic activities: DNA methyltransferases (DNMTs) and Ten-eleven translocation dioxygenases (TETs). In mouse embryonic stem cells (ESCs), de novo DNAme by DNMT3 family enzymes, demethylation by the TET-mediated conversion of 5mC to 5-hydroxymethylation (5hmC), and maintenance of the remaining DNAme by DNMT1 are actively repeated throughout cell cycles, dynamically forming a constant 5mC profile. Nevertheless, the detailed mechanism and physiological significance of this active cyclic DNA modification in mouse ESCs remain unclear. Here by visualizing the localization of DNA modifications on metaphase chromosomes and comparing whole-genome methylation profiles before and after the mid-S phase in ESCs lacking Dnmt1 (1KO ESCs), we demonstrated that in 1KO ESCs, DNMT3-mediated remethylation was interrupted during and after DNA replication. This results in a marked asymmetry in the distribution of 5hmC between sister chromatids at mitosis, with one chromatid being almost no 5hmC. When introduced in 1KO ESCs, the catalytically inactive form of DNMT1 (DNMT1CI) induced an increase in DNAme in pericentric heterochromatin and the DNAme-independent repression of IAPEz, a retrotransposon family, in 1KO ESCs. However, DNMT1CI could not restore the ability of DNMT3 to methylate unmodified dsDNA de novo in S phase in 1KO ESCs. Furthermore, during in vitro differentiation into epiblasts, 1KO ESCs expressing DNMT1CI showed an even stronger tendency to differentiate into the primitive endoderm than 1KO ESCs and were readily reprogrammed into the primitive streak via an epiblast-like cell state, reconfirming the importance of DNMT1 enzymatic activity at the onset of epiblast differentiation. These results indicate a novel function of DNMT1, in which DNMT1 actively regulates the timing and genomic targets of de novo methylation by DNMT3 in an enzymatic activity-dependent and independent manner, respectively.</p>',
'date' => '2022-01-01',
'pmid' => 'https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0262277',
'doi' => '10.1371/journal.pone.0262277',
'modified' => '2022-05-20 09:34:50',
'created' => '2022-05-19 10:41:50',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 1 => array(
'id' => '4045',
'name' => 'Functional role of Tet-mediated RNA hydroxymethylcytosine in mouse ES
cells and during differentiation.',
'authors' => 'Lan, Jie and Rajan, Nicholas and Bizet, Martin and Penning, Audrey and
Singh, Nitesh K and Guallar, Diana and Calonne, Emilie and Li Greci, Andrea
and Bonvin, Elise and Deplus, Rachel and Hsu, Phillip J and Nachtergaele,
Sigrid and Ma, Chengjie and Song, ',
'description' => 'Tet-enzyme-mediated 5-hydroxymethylation of cytosines in DNA plays a
crucial role in mouse embryonic stem cells (ESCs). In RNA also,
5-hydroxymethylcytosine (5hmC) has recently been evidenced, but its
physiological roles are still largely unknown. Here we show the
contribution and function of this mark in mouse ESCs and differentiating
embryoid bodies. Transcriptome-wide mapping in ESCs reveals hundreds of
messenger RNAs marked by 5hmC at sites characterized by a defined unique
consensus sequence and particular features. During differentiation a large
number of transcripts, including many encoding key pluripotency-related
factors (such as Eed and Jarid2), show decreased cytosine
hydroxymethylation. Using Tet-knockout ESCs, we find Tet enzymes to be
partly responsible for deposition of 5hmC in mRNA. A transcriptome-wide
search further reveals mRNA targets to which Tet1 and Tet2 bind, at sites
showing a topology similar to that of 5hmC sites. Tet-mediated RNA
hydroxymethylation is found to reduce the stability of crucial
pluripotency-promoting transcripts. We propose that RNA cytosine
5-hydroxymethylation by Tets is a mark of transcriptome flexibility,
inextricably linked to the balance between pluripotency and lineage
commitment.',
'date' => '2020-10-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/33009383',
'doi' => '10.1038/s41467-020-18729-6',
'modified' => '2021-02-18 10:21:53',
'created' => '2021-02-18 10:21:53',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 2 => array(
'id' => '3660',
'name' => 'Global distribution of DNA hydroxymethylation and DNA methylation in chronic lymphocytic leukemia.',
'authors' => 'Wernig-Zorc S, Yadav MP, Kopparapu PK, Bemark M, Kristjansdottir HL, Andersson PO, Kanduri C, Kanduri M',
'description' => '<p>BACKGROUND: Chronic lymphocytic leukemia (CLL) has been a good model system to understand the functional role of 5-methylcytosine (5-mC) in cancer progression. More recently, an oxidized form of 5-mC, 5-hydroxymethylcytosine (5-hmC) has gained lot of attention as a regulatory epigenetic modification with prognostic and diagnostic implications for several cancers. However, there is no global study exploring the role of 5-hydroxymethylcytosine (5-hmC) levels in CLL. Herein, using mass spectrometry and hMeDIP-sequencing, we analysed the dynamics of 5-hmC during B cell maturation and CLL pathogenesis. RESULTS: We show that naïve B-cells had higher levels of 5-hmC and 5-mC compared to non-class switched and class-switched memory B-cells. We found a significant decrease in global 5-mC levels in CLL patients (n = 15) compared to naïve and memory B cells, with no changes detected between the CLL prognostic groups. On the other hand, global 5-hmC levels of CLL patients were similar to memory B cells and reduced compared to naïve B cells. Interestingly, 5-hmC levels were increased at regulatory regions such as gene-body, CpG island shores and shelves and 5-hmC distribution over the gene-body positively correlated with degree of transcriptional activity. Importantly, CLL samples showed aberrant 5-hmC and 5-mC pattern over gene-body compared to well-defined patterns in normal B-cells. Integrated analysis of 5-hmC and RNA-sequencing from CLL datasets identified three novel oncogenic drivers that could have potential roles in CLL development and progression. CONCLUSIONS: Thus, our study suggests that the global loss of 5-hmC, accompanied by its significant increase at the gene regulatory regions, constitute a novel hallmark of CLL pathogenesis. Our combined analysis of 5-mC and 5-hmC sequencing provided insights into the potential role of 5-hmC in modulating gene expression changes during CLL pathogenesis.</p>',
'date' => '2019-01-07',
'pmid' => 'http://www.pubmed.gov/30616658',
'doi' => '10.1186/s13072‑018‑0252‑7',
'modified' => '2019-07-01 11:46:16',
'created' => '2019-06-21 14:55:31',
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[maximum depth reached]
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),
(int) 3 => array(
'id' => '2992',
'name' => 'Regulation of the DNA Methylation Landscape in Human Somatic Cell Reprogramming by the miR-29 Family',
'authors' => 'Hysolli E et al.',
'description' => 'Reprogramming to pluripotency after overexpression of OCT4, SOX2, KLF4, and MYC is accompanied by global genomic and epigenomic changes. Histone modification and DNA methylation states in induced pluripotent stem cells (iPSCs) have been shown to be highly similar to embryonic stem cells (ESCs). However, epigenetic differences still exist between iPSCs and ESCs. In particular, aberrant DNA methylation states found in iPSCs are a major concern when using iPSCs in a clinical setting. Thus, it is critical to find factors that regulate DNA methylation states in reprogramming. Here, we found that the miR-29 family is an important epigenetic regulator during human somatic cell reprogramming. Our global DNA methylation and hydroxymethylation analysis shows that DNA demethylation is a major event mediated by miR-29a depletion during early reprogramming, and that iPSCs derived from miR-29a depletion are epigenetically closer to ESCs. Our findings uncover an important miRNA-based approach to generate clinically robust iPSCs.',
'date' => '2016-07-12',
'pmid' => 'http://www.ncbi.nlm.nih.gov/pubmed/27373925',
'doi' => '10.1016/j.stemcr.2016.05.014',
'modified' => '2016-08-23 09:57:29',
'created' => '2016-08-23 09:57:29',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 4 => array(
'id' => '2811',
'name' => 'Transcriptome-wide distribution and function of RNA hydroxymethylcytosine',
'authors' => 'Delatte B, Wang F, Ngoc LV, Collignon E, Bonvin E, Deplus R, Calonne E, Hassabi B, Putmans P, Awe S, Wetzel C, Kreher J, Soin R, Creppe C, Limbach PA, Gueydan C, Kruys V, Brehm A, Minakhina S, Defrance M, Steward R, Fuks F.',
'description' => '<p>Hydroxymethylcytosine, well described in DNA, occurs also in RNA. Here, we show that hydroxymethylcytosine preferentially marks polyadenylated RNAs and is deposited by Tet in Drosophila. We map the transcriptome-wide hydroxymethylation landscape, revealing hydroxymethylcytosine in the transcripts of many genes, notably in coding sequences, and identify consensus sites for hydroxymethylation. We found that RNA hydroxymethylation can favor mRNA translation. Tet and hydroxymethylated RNA are found to be most abundant in the Drosophila brain, and Tet-deficient fruitflies suffer impaired brain development, accompanied by decreased RNA hydroxymethylation. This study highlights the distribution, localization, and function of cytosine hydroxymethylation and identifies central roles for this modification in Drosophila.</p>',
'date' => '2016-01-15',
'pmid' => 'http://pubmed.gov/26816380',
'doi' => '10.1126/science.aac5253',
'modified' => '2016-01-28 21:57:55',
'created' => '2016-01-28 21:57:55',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 5 => array(
'id' => '3750',
'name' => 'RNA biochemistry. Transcriptome-wide distribution and function of RNA hydroxymethylcytosine.',
'authors' => 'Delatte B, Wang F, Ngoc LV, Collignon E, Bonvin E, Deplus R, Calonne E, Hassabi B, Putmans P, Awe S, Wetzel C, Kreher J, Soin R, Creppe C, Limbach PA, Gueydan C, Kruys V, Brehm A, Minakhina S, Defrance M, Steward R, Fuks F',
'description' => '<p>Hydroxymethylcytosine, well described in DNA, occurs also in RNA. Here, we show that hydroxymethylcytosine preferentially marks polyadenylated RNAs and is deposited by Tet in Drosophila. We map the transcriptome-wide hydroxymethylation landscape, revealing hydroxymethylcytosine in the transcripts of many genes, notably in coding sequences, and identify consensus sites for hydroxymethylation. We found that RNA hydroxymethylation can favor mRNA translation. Tet and hydroxymethylated RNA are found to be most abundant in the Drosophila brain, and Tet-deficient fruitflies suffer impaired brain development, accompanied by decreased RNA hydroxymethylation. This study highlights the distribution, localization, and function of cytosine hydroxymethylation and identifies central roles for this modification in Drosophila.</p>',
'date' => '2016-01-15',
'pmid' => 'http://www.pubmed.org/26816380',
'doi' => '10.1126/science.aac5253',
'modified' => '2019-10-03 12:29:05',
'created' => '2019-10-02 16:16:55',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 6 => array(
'id' => '2613',
'name' => 'CpG signalling, H2A.Z/H3 acetylation and microRNA-mediated deferred self-attenuation orchestrate foetal NOS3 expression.',
'authors' => 'Postberg J, Kanders M, Forcob S, Willems R, Orth V, Hensel KO, Weil PP, Wirth S, Jenke AC',
'description' => 'BACKGROUND: An adverse intrauterine environment leads to permanent physiological changes including vascular tone regulation, potentially influencing the risk for adult vascular diseases. We therefore aimed to monitor responsive NOS3 expression in human umbilical artery endothelial cells (HUAEC) and to study the underlying epigenetic signatures involved in its regulation. RESULTS: NOS3 and STAT3 mRNA levels were elevated in HUAEC of patients who suffered from placental insufficiency. 5-hydroxymethylcytosine, H3K9ac and Histone 2A (H2A).Zac at the NOS3 transcription start site directly correlated with NOS3 mRNA levels. Concomitantly, we observed entangled histone acetylation patterns and NOS3 response upon hypoxic conditions in vitro. Knock-down of either NOS3 or STAT3 by RNAi provided evidence for a functional NOS3/STAT3 relationship. Moreover, we recognized massive turnover of Stat3 at a discrete binding site in the NOS3 promoter. Interestingly, induced hyperacetylation resulted in short-termed increase of NOS3 mRNA followed by deferred decrease indicating that NOS3 expression could become self-attenuated by co-expressed intronic 27 nt-ncRNA. Reporter assay results and phylogenetic analyses enabled us to propose a novel model for STAT3-3'-UTR targeting by this 27-nt-ncRNA. CONCLUSIONS: An adverse intrauterine environment leads to adaptive changes of NOS3 expression. Apparently, a rapid NOS3 self-limiting response upon ectopic triggers co-exists with longer termed expression changes in response to placental insufficiency involving differential epigenetic signatures. Their persistence might contribute to impaired vascular endothelial response and consequently increase the risk of cardiovascular disease later in life.',
'date' => '2015-01-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/25699114',
'doi' => '',
'modified' => '2015-07-24 15:39:05',
'created' => '2015-07-24 15:39:05',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 7 => array(
'id' => '2348',
'name' => 'White matter tract and glial-associated changes in 5-hydroxymethylcytosine following chronic cerebral hypoperfusion.',
'authors' => 'Tsenkina Y, Ruzov A, Gliddon C, Horsburgh K, De Sousa PA',
'description' => 'White matter abnormalities due to age-related cerebrovascular alterations is a common pathological hallmark associated with functional impairment in the elderly which has been modeled in chronically hypoperfused mice. 5-Methylcytosine (5mC) and its oxidized derivative 5-hydroxymethylcytosine (5hmC) are DNA modifications that have been recently linked with age-related neurodegeneration and cerebrovascular pathology. Here we conducted a pilot investigation of whether chronic cerebral hypoperfusion might affect genomic distribution of these modifications and/ or a Ten-Eleven Translocation protein 2 (TET2) which catalyses hydroxymethylation in white and grey matter regions of this animal model. Immunohistochemical evaluation of sham and chronically hypoperfused mice a month after surgery revealed significant (p<0.05) increases in the proportion of 5hmC positive cells, Iba1 positive inflammatory microglia, and NG2 positive oligodendroglial progenitors in the hypoperfused corpus callosum. In the same white matter tract there was an absence of hypoperfusion-induced alterations in the proportion of 5mC, TET2 positive cells and CC1 positive mature oligodrendrocytes. Correlation analysis across animals within both treatment groups demonstrated a significant association of the elevated 5hmC levels with increases in the proportion of inflammatory microglia only (p=0.01) in the corpus callosum. In vitro studies revealed that 5hmC is lost during oligodendroglial maturation but not microglial activation. Additionally, TET1, TET2, and TET3 protein levels showed dynamic alterations during oligodendroglial development and following oxidative stress in vitro. Our study suggests that 5hmC exhibits white matter tract and cell type specific dynamics following chronic cerebral hypoperfusion in mice.',
'date' => '2014-10-10',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/25305569',
'doi' => '',
'modified' => '2015-07-24 15:39:04',
'created' => '2015-07-24 15:39:04',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 8 => array(
'id' => '994',
'name' => 'Tet2 Facilitates the Derepression of Myeloid Target Genes during CEBPα-Induced Transdifferentiation of Pre-B Cells.',
'authors' => 'Kallin EM, Rodríguez-Ubreva J, Christensen J, Cimmino L, Aifantis I, Helin K, Ballestar E, Graf T',
'description' => 'The methylcytosine hydroxylase Tet2 has been implicated in hematopoietic differentiation and the formation of myeloid malignancies when mutated. An ideal system to study the role of Tet2 in myelopoeisis is CEBPα-induced transdifferentiation of pre-B cells into macrophages. Here we found that CEBPα binds to upstream regions of Tet2 and that the gene becomes activated. Tet2 knockdowns impaired the upregulation of macrophage markers as well as phagocytic capacity, suggesting that the enzyme is required for both early and late stage myeloid differentiation. A slightly weaker effect was seen in primary cells with a Tet2 ablation. Expression arrays of transdifferentiating cells with Tet2 knockdowns permitted the identification of a small subset of myeloid genes whose upregulation was blunted. Activation of these target genes was accompanied by rapid increases of promoter hydroxy-methylation. Our observations indicate that Tet2 helps CEBPα rapidly derepress myeloid genes during the conversion of pre-B cells into macrophages.',
'date' => '2012-09-12',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/22981865',
'doi' => '',
'modified' => '2015-07-24 15:38:59',
'created' => '2015-07-24 15:38:59',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 9 => array(
'id' => '149',
'name' => 'Lineage-specific distribution of high levels of genomic 5-hydroxymethylcytosine in mammalian development.',
'authors' => 'Ruzov A, Tsenkina Y, Serio A, Dudnakova T, Fletcher J, Bai Y, Chebotareva T, Pells S, Hannoun Z, Sullivan G, Chandran S, Hay DC, Bradley M, Wilmut I, De Sousa P',
'description' => 'Methylation of cytosine is a DNA modification associated with gene repression. Recently, a novel cytosine modification, 5-hydroxymethylcytosine (5-hmC) has been discovered. Here we examine 5-hmC distribution during mammalian development and in cellular systems, and show that the developmental dynamics of 5-hmC are different from those of 5-methylcytosine (5-mC); in particular 5-hmC is enriched in embryonic contexts compared to adult tissues. A detectable 5-hmC signal appears in pre-implantation development starting at the zygote stage, where the paternal genome is subjected to a genome-wide hydroxylation of 5-mC, which precisely coincides with the loss of the 5-mC signal in the paternal pronucleus. Levels of 5-hmC are high in cells of the inner cell mass in blastocysts, and the modification colocalises with nestin-expressing cell populations in mouse post-implantation embryos. Compared to other adult mammalian organs, 5-hmC is strongly enriched in bone marrow and brain, wherein high 5-hmC content is a feature of both neuronal progenitors and post-mitotic neurons. We show that high levels of 5-hmC are not only present in mouse and human embryonic stem cells (ESCs) and lost during differentiation, as has been reported previously, but also reappear during the generation of induced pluripotent stem cells; thus 5-hmC enrichment correlates with a pluripotent cell state. Our findings suggest that apart from the cells of neuronal lineages, high levels of genomic 5-hmC are an epigenetic feature of embryonic cell populations and cellular pluri- and multi-lineage potency. To our knowledge, 5-hmC represents the first epigenetic modification of DNA discovered whose enrichment is so cell-type specific.',
'date' => '2011-09-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/21747414',
'doi' => '',
'modified' => '2015-07-24 15:38:57',
'created' => '2015-07-24 15:38:57',
'ProductsPublication' => array(
[maximum depth reached]
)
),
(int) 10 => array(
'id' => '361',
'name' => 'Genome-wide analysis of 5-hydroxymethylcytosine distribution reveals its dual function in transcriptional regulation in mouse embryonic stem cells.',
'authors' => 'Wu H, D'Alessio AC, Ito S, Wang Z, Cui K, Zhao K, Sun YE, Zhang Y',
'description' => 'Recent studies have demonstrated that the Ten-eleven translocation (Tet) family proteins can enzymatically convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). While 5mC has been studied extensively, little is known about the distribution and function of 5hmC. Here we present a genome-wide profile of 5hmC in mouse embryonic stem (ES) cells. A combined analysis of global 5hmC distribution and gene expression profile in wild-type and Tet1-depleted ES cells suggests that 5hmC is enriched at both gene bodies of actively transcribed genes and extended promoter regions of Polycomb-repressed developmental regulators. Thus, our study reveals the first genome-wide 5hmC distribution in pluripotent stem cells, and supports its dual function in regulating gene expression.',
'date' => '2011-04-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/21460036',
'doi' => '',
'modified' => '2015-07-24 15:38:57',
'created' => '2015-07-24 15:38:57',
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[maximum depth reached]
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'antibody_id' => '59',
'name' => '5-hydroxymethylcytosine (5-hmC) Antibody (rat) ',
'description' => '<p>5<span>-hmC is a DNA modification which results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. Preliminary results indicate that 5-hmC may have important roles distinct from 5-methylcytosine (5-mC). Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics.</span></p>',
'label1' => 'Validation Data',
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<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig1.png" alt="hMeDIP" style="display: block; margin-left: auto; margin-right: auto;" /></p>
</div>
<div class="small-6 columns">
<p><small><strong>Figure 1. Hydroxymethylated DNA IP results obtained with our hMeDIP kit (Cat. No. AF-104-0016)</strong><br /> Hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit. This kit includes: the monoclonal antibody against 5-hydroxymethylcytosine (Cat. No. MAb-633HMC-050), 5-hmC, 5-mC & cytosine DNA standards & Rat IgG (Cat. No. AF-105-0025). The DNA was prepared with the GenDNA module and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. 1 μg of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP’d material has been analysed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control • with Rat IgG as negative control (0.06%, almost no recovery) • with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control • with Rat IgG as negative control (0.03%, almost no recovery) • with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control • with Rat IgG as negative control (0.04%, almost no recovery) • with 5-hmC (97.60% recovery, almost full recovery) These results clearly demonstrate the high specificity and efficiency of the 5-hydroxymethylcytosine monoclonal antibody.</small></p>
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<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig2.png" alt="ELISA" style="display: block; margin-left: auto; margin-right: auto;" width="375" height="274" /></p>
</div>
<div class="small-6 columns">
<p><small><strong>Figure 2. Determination of the 5-hmC rat monoclonal antibody titer</strong><br />To determine the titer, an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody directed against 5-hmC (Cat No. MAb-633HMC-050, MAb-633HMC-100) in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.</small></p>
</div>
</div>
<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig3.png" alt="Dot blot" style="display: block; margin-left: auto; margin-right: auto;" width="190" height="192" /></p>
</div>
<div class="small-6 columns">
<p><small><strong>Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls</strong><br />Figure 3A: Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.</small></p>
</div>
</div>
<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig4.png" style="display: block; margin-left: auto; margin-right: auto;" width="115" height="232" /></p>
</div>
<div class="small-6 columns">
<p><small><strong>Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls</strong><br />200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode « 5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.</small></p>
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'label2' => 'Target description',
'info2' => '<p>5-hydroxymethylcytosine (5-hmC) has been recently discovered in mammalian DNA. This results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. So far, the 5-hmC bases have been identified in Purkinje neurons, in granule cells and embryonic stem cells where they are present at high levels (up to 0,6% of total nucleotides in Purkinje cells).</p>
<p>Preliminary results indicate that 5-hmC may have important roles distinct from 5-mC. Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics : 5-hydroxymethylcytosine may well represent a new pathway to demethylate DNA involving a repair mechanism converting 5-hmC to cytosine and, as such open up entirely new perspectives in epigenetic studies.</p>
<p>Due to the structural similarity between 5-mC and 5-hmC, these bases are experimentally almost indistinguishable. Recent articles demonstrated that the most common approaches (e.g. enzymatic approaches, bisulfite sequencing) do not account for 5-hmC. The development of the affinity-based technologies appears to be the most powerful way to differentially and specifically enrich 5-mC and 5-hmC sequences. The results shown here illustrate the use of this unique monoclonal antibody against 5-hydroxymethylcytosine that has been fully validated in various technologies.</p>',
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'meta_title' => '5-hydroxymethylcytosine (5-hmC) Monoclonal Antibody (rat) | Diagenode',
'meta_keywords' => '5-hydroxymethylcytosine,5-hmC, 5-mC,monoclonal antibody ,Diagenode',
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'id' => '2032',
'antibody_id' => '59',
'name' => '5-hydroxymethylcytosine (5-hmC) Antibody (rat) (sample size)',
'description' => '<p><span>5-hmC is a DNA modification which results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. Preliminary results indicate that 5-hmC may have important roles distinct from 5-methylcytosine (5-mC). Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics.</span></p>',
'label1' => 'Validation Data',
'info1' => '<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig1.png" alt="hMeDIP" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 1. Hydroxymethylated DNA IP results obtained with our hMeDIP kit (Cat. No. AF-104-0016)</strong><br /> Hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit. This kit includes: the monoclonal antibody against 5-hydroxymethylcytosine (Cat. No. MAb-633HMC-050), 5-hmC, 5-mC & cytosine DNA standards & Rat IgG (Cat. No. AF-105-0025). The DNA was prepared with the GenDNA module and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. 1 μg of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP’d material has been analysed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control • with Rat IgG as negative control (0.06%, almost no recovery) • with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control • with Rat IgG as negative control (0.03%, almost no recovery) • with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control • with Rat IgG as negative control (0.04%, almost no recovery) • with 5-hmC (97.60% recovery, almost full recovery) These results clearly demonstrate the high specificity and efficiency of the 5-hydroxymethylcytosine monoclonal antibody.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig2.png" alt="ELISA" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 2. Determination of the 5-hmC rat monoclonal antibody titer</strong><br /> To determine the titer, an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody directed against 5-hmC (Cat No. MAb-633HMC-050, MAb-633HMC-100) in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig3.png" alt="Dot blot" style="display: block; margin-left: auto; margin-right: auto;" width="200" height="200" /></p>
<p><strong>Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls</strong><br /> Figure 3A: Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig4.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls</strong><br /> 200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode « 5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.</p>',
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'name' => '5-hydroxymethylcytosine (5-hmC) Antibody (rat) ',
'description' => '<p>5<span>-hmC is a DNA modification which results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. Preliminary results indicate that 5-hmC may have important roles distinct from 5-methylcytosine (5-mC). Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics.</span></p>',
'label1' => 'Validation Data',
'info1' => '<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig1.png" alt="hMeDIP" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 1. Hydroxymethylated DNA IP results obtained with our hMeDIP kit (Cat. No. AF-104-0016)</strong><br /> Hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit. This kit includes: the monoclonal antibody against 5-hydroxymethylcytosine (Cat. No. MAb-633HMC-050), 5-hmC, 5-mC & cytosine DNA standards & Rat IgG (Cat. No. AF-105-0025). The DNA was prepared with the GenDNA module and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. 1 μg of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP’d material has been analysed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control • with Rat IgG as negative control (0.06%, almost no recovery) • with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control • with Rat IgG as negative control (0.03%, almost no recovery) • with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control • with Rat IgG as negative control (0.04%, almost no recovery) • with 5-hmC (97.60% recovery, almost full recovery) These results clearly demonstrate the high specificity and efficiency of the 5-hydroxymethylcytosine monoclonal antibody.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig2.png" alt="ELISA" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 2. Determination of the 5-hmC rat monoclonal antibody titer</strong><br /> To determine the titer, an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody directed against 5-hmC (Cat No. MAb-633HMC-050, MAb-633HMC-100) in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig3.png" alt="Dot blot" style="display: block; margin-left: auto; margin-right: auto;" width="200" height="200" /></p>
<p><strong>Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls</strong><br /> Figure 3A: Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig4.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls</strong><br /> 200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode « 5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.</p>',
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<h6 style="height:60px">hMeDIP kit x16 (monoclonal mouse antibody)</h6>
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<p><span>The hMeDIP kit is designed for enrichment of hydroxymethylated DNA from fragmented genomic DNA<span><span> </span>samples for use in genome-wide methylation analysis. It features</span></span><span> a highly specific monoclonal antibody against </span>5-hydroxymethylcytosine (5-hmC) for the immunoprecipitation of hydroxymethylated DNA<span>. It includes control DNA and primers to assess the effiency of the assay. </span>Performing hydroxymethylation profiling with the hMeDIP kit is fast, reliable and highly specific.</p>
<p><em>Looking for hMeDIP-seq protocol? <a href="https://go.diagenode.com/l/928883/2022-01-07/2m1ht" target="_blank" title="Contact us">Contact us</a></em></p>
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<p><strong>Figure 1. Hydroxymethylated DNA IP results obtained with our hMeDIP kit (Cat. No. AF-104-0016)</strong><br /> Hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit. This kit includes: the monoclonal antibody against 5-hydroxymethylcytosine (Cat. No. MAb-633HMC-050), 5-hmC, 5-mC & cytosine DNA standards & Rat IgG (Cat. No. AF-105-0025). The DNA was prepared with the GenDNA module and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. 1 μg of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP’d material has been analysed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control • with Rat IgG as negative control (0.06%, almost no recovery) • with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control • with Rat IgG as negative control (0.03%, almost no recovery) • with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control • with Rat IgG as negative control (0.04%, almost no recovery) • with 5-hmC (97.60% recovery, almost full recovery) These results clearly demonstrate the high specificity and efficiency of the 5-hydroxymethylcytosine monoclonal antibody.</p>
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<p><strong>Figure 2. Determination of the 5-hmC rat monoclonal antibody titer</strong><br /> To determine the titer, an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody directed against 5-hmC (Cat No. MAb-633HMC-050, MAb-633HMC-100) in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig3.png" alt="Dot blot" style="display: block; margin-left: auto; margin-right: auto;" width="200" height="200" /></p>
<p><strong>Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls</strong><br /> Figure 3A: Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig4.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls</strong><br /> 200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode « 5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.</p>',
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'description' => '<p>Cytosine hydroxymethylation was recently discovered as an important epigenetic mechanism. This cytosine base modification results from the enzymatic conversion of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC) by the TET family of oxygenases. Though the precise role of 5-hmC is the subject of intense research and debate, early studies strongly indicate that it is also involved in gene regulation and in numerous important biological processes including embryonic development, cellular differentiation, stem cell reprogramming and carcinogenesis.</p>
<p>The study of 5-hmC has long been limited due to the lack of high quality, validated tools and technologies that discriminate hydroxymethylation from methylation in regulating gene expression. The use of highly specific antibodies against 5-hmC for the immunoprecipitation of hydroxymethylated DNA offers a reliable solution for hydroxymethylation profiling.</p>
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'description' => 'Recent studies have demonstrated that the Ten-eleven translocation (Tet) family proteins can enzymatically convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). While 5mC has been studied extensively, little is known about the distribution and function of 5hmC. Here we present a genome-wide profile of 5hmC in mouse embryonic stem (ES) cells. A combined analysis of global 5hmC distribution and gene expression profile in wild-type and Tet1-depleted ES cells suggests that 5hmC is enriched at both gene bodies of actively transcribed genes and extended promoter regions of Polycomb-repressed developmental regulators. Thus, our study reveals the first genome-wide 5hmC distribution in pluripotent stem cells, and supports its dual function in regulating gene expression.',
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<p><strong>Figure 1. Hydroxymethylated DNA IP results obtained with our hMeDIP kit (Cat. No. AF-104-0016)</strong><br /> Hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit. This kit includes: the monoclonal antibody against 5-hydroxymethylcytosine (Cat. No. MAb-633HMC-050), 5-hmC, 5-mC & cytosine DNA standards & Rat IgG (Cat. No. AF-105-0025). The DNA was prepared with the GenDNA module and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. 1 μg of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP’d material has been analysed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control • with Rat IgG as negative control (0.06%, almost no recovery) • with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control • with Rat IgG as negative control (0.03%, almost no recovery) • with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control • with Rat IgG as negative control (0.04%, almost no recovery) • with 5-hmC (97.60% recovery, almost full recovery) These results clearly demonstrate the high specificity and efficiency of the 5-hydroxymethylcytosine monoclonal antibody.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig2.png" alt="ELISA" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 2. Determination of the 5-hmC rat monoclonal antibody titer</strong><br /> To determine the titer, an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody directed against 5-hmC (Cat No. MAb-633HMC-050, MAb-633HMC-100) in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig3.png" alt="Dot blot" style="display: block; margin-left: auto; margin-right: auto;" width="200" height="200" /></p>
<p><strong>Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls</strong><br /> Figure 3A: Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig4.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls</strong><br /> 200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode « 5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.</p>',
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<p><strong>Figure 1. Hydroxymethylated DNA IP results obtained with our hMeDIP kit (Cat. No. AF-104-0016)</strong><br /> Hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit. This kit includes: the monoclonal antibody against 5-hydroxymethylcytosine (Cat. No. MAb-633HMC-050), 5-hmC, 5-mC & cytosine DNA standards & Rat IgG (Cat. No. AF-105-0025). The DNA was prepared with the GenDNA module and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. 1 μg of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP’d material has been analysed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control • with Rat IgG as negative control (0.06%, almost no recovery) • with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control • with Rat IgG as negative control (0.03%, almost no recovery) • with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control • with Rat IgG as negative control (0.04%, almost no recovery) • with 5-hmC (97.60% recovery, almost full recovery) These results clearly demonstrate the high specificity and efficiency of the 5-hydroxymethylcytosine monoclonal antibody.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig2.png" alt="ELISA" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 2. Determination of the 5-hmC rat monoclonal antibody titer</strong><br /> To determine the titer, an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody directed against 5-hmC (Cat No. MAb-633HMC-050, MAb-633HMC-100) in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig3.png" alt="Dot blot" style="display: block; margin-left: auto; margin-right: auto;" width="200" height="200" /></p>
<p><strong>Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls</strong><br /> Figure 3A: Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig4.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls</strong><br /> 200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode « 5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.</p>',
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<p>Preliminary results indicate that 5-hmC may have important roles distinct from 5-mC. Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics : 5-hydroxymethylcytosine may well represent a new pathway to demethylate DNA involving a repair mechanism converting 5-hmC to cytosine and, as such open up entirely new perspectives in epigenetic studies.</p>
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Preliminary results indicate that 5-hmC may have important roles distinct from 5-mC. Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics : 5-hydroxymethylcytosine may well represent a new pathway to demethylate DNA involving a repair mechanism converting 5-hmC to cytosine and, as such open up entirely new perspectives in epigenetic studies.
Due to the structural similarity between 5-mC and 5-hmC, these bases are experimentally almost indistinguishable. Recent articles demonstrated that the most common approaches (e.g. enzymatic approaches, bisulfite sequencing) do not account for 5-hmC. The development of the affinity-based technologies appears to be the most powerful way to differentially and specifically enrich 5-mC and 5-hmC sequences. The results shown here illustrate the use of this unique monoclonal antibody against 5-hydroxymethylcytosine that has been fully validated in various technologies.',
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<td>Fig 1</td>
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<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig1.png" alt="hMeDIP" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<p><small><strong>Figure 1. Hydroxymethylated DNA IP results obtained with our hMeDIP kit (Cat. No. AF-104-0016)</strong><br /> Hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit. This kit includes: the monoclonal antibody against 5-hydroxymethylcytosine (Cat. No. MAb-633HMC-050), 5-hmC, 5-mC & cytosine DNA standards & Rat IgG (Cat. No. AF-105-0025). The DNA was prepared with the GenDNA module and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. 1 μg of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP’d material has been analysed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control • with Rat IgG as negative control (0.06%, almost no recovery) • with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control • with Rat IgG as negative control (0.03%, almost no recovery) • with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control • with Rat IgG as negative control (0.04%, almost no recovery) • with 5-hmC (97.60% recovery, almost full recovery) These results clearly demonstrate the high specificity and efficiency of the 5-hydroxymethylcytosine monoclonal antibody.</small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig2.png" alt="ELISA" style="display: block; margin-left: auto; margin-right: auto;" width="375" height="274" /></p>
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<p><small><strong>Figure 2. Determination of the 5-hmC rat monoclonal antibody titer</strong><br />To determine the titer, an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody directed against 5-hmC (Cat No. MAb-633HMC-050, MAb-633HMC-100) in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.</small></p>
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<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig3.png" alt="Dot blot" style="display: block; margin-left: auto; margin-right: auto;" width="190" height="192" /></p>
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<p><small><strong>Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls</strong><br />Figure 3A: Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.</small></p>
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<p><small><strong>Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls</strong><br />200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode « 5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.</small></p>
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<p>Preliminary results indicate that 5-hmC may have important roles distinct from 5-mC. Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics : 5-hydroxymethylcytosine may well represent a new pathway to demethylate DNA involving a repair mechanism converting 5-hmC to cytosine and, as such open up entirely new perspectives in epigenetic studies.</p>
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<li><strong>High capture efficiency</strong></li>
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<h3>MBD-seq allows for detection of genomic regions with different CpG density</h3>
<p><img src="https://www.diagenode.com/img/product/kits/mbd_results1.png" alt="MBD-sequencing results have been validated by bisulfite sequencing" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>F</strong><strong>igure 1.</strong><span> </span>Using the MBD approach, two methylated regions were detected in different elution fractions according to their methylated CpG density (A). Low, Medium and High refer to the sequenced DNA from different elution fractions with increasing salt concentration. Methylated patterns of these two different methylated regions were validated by bisulfite conversion assay (B).<br /><strong></strong></p>
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<li><strong>On-day protocol</strong></li>
<li><strong>NGS compatibility</strong></li>
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<h3>MBD-seq allows for detection of genomic regions with different CpG density</h3>
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<p><strong></strong><strong>F</strong><strong>igure 1.</strong> Using the MBD approach, two methylated regions were detected in different elution fractions according to their methylated CpG density (A). Low, Medium and High refer to the sequenced DNA from different elution fractions with increasing salt concentration. Methylated patterns of these two different methylated regions were validated by bisulfite conversion assay (B).</p>',
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<p style="text-align: center;"><strong>Make your Bisulfite conversion now in only 60 minutes !</strong></p>
<p>Diagenode's Premium Bisulfite Kit rapidly converts DNA through bisulfite treatment. Our conversion reagent is added directly to DNA, requires no intermediate steps, and results in high yields of DNA ready for downstream analysis methods including PCR and Next-Generation Sequencing.</p>',
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<p><span>The hMeDIP kit is designed for enrichment of hydroxymethylated DNA from fragmented genomic DNA<span><span> </span>samples for use in genome-wide methylation analysis. It features</span></span><span> a highly specific monoclonal antibody against </span>5-hydroxymethylcytosine (5-hmC) for the immunoprecipitation of hydroxymethylated DNA<span>. It includes control DNA and primers to assess the effiency of the assay. </span>Performing hydroxymethylation profiling with the hMeDIP kit is fast, reliable and highly specific.</p>
<p><em>Looking for hMeDIP-seq protocol? <a href="https://go.diagenode.com/l/928883/2022-01-07/2m1ht" target="_blank" title="Contact us">Contact us</a></em></p>
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<li>Including control DNA and primers to <span>monitor the efficiency of the assay</span>
<ul style="list-style-type: circle;">
<li>hmeDNA and unmethylated DNA sequences and primer pairs</li>
<li>Mouse primer pairs against Sfi1 targeting hydroxymethylated gene in mouse</li>
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<li>Improved single-tube, magnetic bead-based protocol</li>
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'description' => '<p><span style="font-weight: 400;">T</span><span style="font-weight: 400;">he pattern of <strong>DNA modifications</strong> is critical for genome stability and the control of gene expression in the cell. Methylation of 5-cytosine (5-mC), one of the best-studied epigenetic marks, is carried out by the <strong>DNA methyltransferases</strong> DNMT3A and B and DNMT1. DNMT3A and DNMT3B are responsible for </span><i><span style="font-weight: 400;">de novo</span></i><span style="font-weight: 400;"> DNA methylation, whereas DNMT1 maintains existing methylation. 5-mC undergoes active demethylation which is performed by the <strong>Ten-Eleven Translocation</strong> (TET) familly of DNA hydroxylases. The latter consists of 3 members TET1, 2 and 3. All 3 members catalyze the conversion of <strong>5-methylcytosine</strong> (5-mC) into <strong>5-hydroxymethylcytosine</strong> (5-hmC), and further into <strong>5-formylcytosine</strong> (5-fC) and <strong>5-carboxycytosine</strong> (5-caC). 5-fC and 5-caC can be converted to unmodified cytosine by <strong>Thymine DNA Glycosylase</strong> (TDG). It is not yet clear if 5-hmC, 5-fC and 5-caC have specific functions or are simply intermediates in the demethylation of 5-mC.</span></p>
<p><span style="font-weight: 400;">DNA methylation is generally considered as a repressive mark and is usually associated with gene silencing. It is essential that the balance between DNA methylation and demethylation is precisely maintained. Dysregulation of DNA methylation may lead to many different human diseases and is often observed in cancer cells.</span></p>
<p><span style="font-weight: 400;">Diagenode offers highly validated antibodies against different proteins involved in DNA modifications as well as against the modified bases allowing the study of all steps and intermediates in the DNA methylation/demethylation pathway:</span></p>
<p><img src="https://www.diagenode.com/img/categories/antibodies/dna-methylation.jpg" height="599" width="816" /></p>
<p><strong>Diagenode exclusively sources the original 5-methylcytosine monoclonal antibody (clone 33D3).</strong></p>
<p>Check out the list below to see all proposed antibodies for DNA modifications.</p>
<p>Diagenode’s highly validated antibodies:</p>
<ul>
<li>Highly sensitive and specific</li>
<li>Cost-effective (requires less antibody per reaction)</li>
<li>Batch-specific data is available on the website</li>
<li>Expert technical support</li>
<li>Sample sizes available</li>
<li>100% satisfaction guarantee</li>
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<p><span style="font-weight: 400;">Diagenode’s highly validated antibodies:</span></p>
<ul>
<li>Highly sensitive and specific</li>
<li>Cost-effective (requires less antibody per reaction)</li>
<li>Batch-specific data is available on the website</li>
<li>Expert technical support</li>
<li>Sample sizes available</li>
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'description' => '<p>5<span>-hmC is a DNA modification which results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. Preliminary results indicate that 5-hmC may have important roles distinct from 5-methylcytosine (5-mC). Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics.</span></p>',
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'description' => '<p>Cytosine hydroxymethylation was recently discovered as an important epigenetic mechanism. This cytosine base modification results from the enzymatic conversion of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC) by the TET family of oxygenases. Though the precise role of 5-hmC is the subject of intense research and debate, early studies strongly indicate that it is also involved in gene regulation and in numerous important biological processes including embryonic development, cellular differentiation, stem cell reprogramming and carcinogenesis.</p>
<p>The study of 5-hmC has long been limited due to the lack of high quality, validated tools and technologies that discriminate hydroxymethylation from methylation in regulating gene expression. The use of highly specific antibodies against 5-hmC for the immunoprecipitation of hydroxymethylated DNA offers a reliable solution for hydroxymethylation profiling.</p>
<p></p>',
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<div class="section">
<div class="layoutArea">
<div class="column">
<p><span> </span></p>
</div>
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</div>
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'name' => 'DNMT1 regulates the timing of DNA methylation by DNMT3 in anenzymatic activity-dependent manner in mouse embryonic stem cells.',
'authors' => 'Ito Takamasa et al.',
'description' => '<p>DNA methylation (DNAme; 5-methylcytosine, 5mC) plays an essential role in mammalian development, and the 5mC profile is regulated by a balance of opposing enzymatic activities: DNA methyltransferases (DNMTs) and Ten-eleven translocation dioxygenases (TETs). In mouse embryonic stem cells (ESCs), de novo DNAme by DNMT3 family enzymes, demethylation by the TET-mediated conversion of 5mC to 5-hydroxymethylation (5hmC), and maintenance of the remaining DNAme by DNMT1 are actively repeated throughout cell cycles, dynamically forming a constant 5mC profile. Nevertheless, the detailed mechanism and physiological significance of this active cyclic DNA modification in mouse ESCs remain unclear. Here by visualizing the localization of DNA modifications on metaphase chromosomes and comparing whole-genome methylation profiles before and after the mid-S phase in ESCs lacking Dnmt1 (1KO ESCs), we demonstrated that in 1KO ESCs, DNMT3-mediated remethylation was interrupted during and after DNA replication. This results in a marked asymmetry in the distribution of 5hmC between sister chromatids at mitosis, with one chromatid being almost no 5hmC. When introduced in 1KO ESCs, the catalytically inactive form of DNMT1 (DNMT1CI) induced an increase in DNAme in pericentric heterochromatin and the DNAme-independent repression of IAPEz, a retrotransposon family, in 1KO ESCs. However, DNMT1CI could not restore the ability of DNMT3 to methylate unmodified dsDNA de novo in S phase in 1KO ESCs. Furthermore, during in vitro differentiation into epiblasts, 1KO ESCs expressing DNMT1CI showed an even stronger tendency to differentiate into the primitive endoderm than 1KO ESCs and were readily reprogrammed into the primitive streak via an epiblast-like cell state, reconfirming the importance of DNMT1 enzymatic activity at the onset of epiblast differentiation. These results indicate a novel function of DNMT1, in which DNMT1 actively regulates the timing and genomic targets of de novo methylation by DNMT3 in an enzymatic activity-dependent and independent manner, respectively.</p>',
'date' => '2022-01-01',
'pmid' => 'https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0262277',
'doi' => '10.1371/journal.pone.0262277',
'modified' => '2022-05-20 09:34:50',
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(int) 1 => array(
'id' => '4045',
'name' => 'Functional role of Tet-mediated RNA hydroxymethylcytosine in mouse ES
cells and during differentiation.',
'authors' => 'Lan, Jie and Rajan, Nicholas and Bizet, Martin and Penning, Audrey and
Singh, Nitesh K and Guallar, Diana and Calonne, Emilie and Li Greci, Andrea
and Bonvin, Elise and Deplus, Rachel and Hsu, Phillip J and Nachtergaele,
Sigrid and Ma, Chengjie and Song, ',
'description' => 'Tet-enzyme-mediated 5-hydroxymethylation of cytosines in DNA plays a
crucial role in mouse embryonic stem cells (ESCs). In RNA also,
5-hydroxymethylcytosine (5hmC) has recently been evidenced, but its
physiological roles are still largely unknown. Here we show the
contribution and function of this mark in mouse ESCs and differentiating
embryoid bodies. Transcriptome-wide mapping in ESCs reveals hundreds of
messenger RNAs marked by 5hmC at sites characterized by a defined unique
consensus sequence and particular features. During differentiation a large
number of transcripts, including many encoding key pluripotency-related
factors (such as Eed and Jarid2), show decreased cytosine
hydroxymethylation. Using Tet-knockout ESCs, we find Tet enzymes to be
partly responsible for deposition of 5hmC in mRNA. A transcriptome-wide
search further reveals mRNA targets to which Tet1 and Tet2 bind, at sites
showing a topology similar to that of 5hmC sites. Tet-mediated RNA
hydroxymethylation is found to reduce the stability of crucial
pluripotency-promoting transcripts. We propose that RNA cytosine
5-hydroxymethylation by Tets is a mark of transcriptome flexibility,
inextricably linked to the balance between pluripotency and lineage
commitment.',
'date' => '2020-10-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/33009383',
'doi' => '10.1038/s41467-020-18729-6',
'modified' => '2021-02-18 10:21:53',
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(int) 2 => array(
'id' => '3660',
'name' => 'Global distribution of DNA hydroxymethylation and DNA methylation in chronic lymphocytic leukemia.',
'authors' => 'Wernig-Zorc S, Yadav MP, Kopparapu PK, Bemark M, Kristjansdottir HL, Andersson PO, Kanduri C, Kanduri M',
'description' => '<p>BACKGROUND: Chronic lymphocytic leukemia (CLL) has been a good model system to understand the functional role of 5-methylcytosine (5-mC) in cancer progression. More recently, an oxidized form of 5-mC, 5-hydroxymethylcytosine (5-hmC) has gained lot of attention as a regulatory epigenetic modification with prognostic and diagnostic implications for several cancers. However, there is no global study exploring the role of 5-hydroxymethylcytosine (5-hmC) levels in CLL. Herein, using mass spectrometry and hMeDIP-sequencing, we analysed the dynamics of 5-hmC during B cell maturation and CLL pathogenesis. RESULTS: We show that naïve B-cells had higher levels of 5-hmC and 5-mC compared to non-class switched and class-switched memory B-cells. We found a significant decrease in global 5-mC levels in CLL patients (n = 15) compared to naïve and memory B cells, with no changes detected between the CLL prognostic groups. On the other hand, global 5-hmC levels of CLL patients were similar to memory B cells and reduced compared to naïve B cells. Interestingly, 5-hmC levels were increased at regulatory regions such as gene-body, CpG island shores and shelves and 5-hmC distribution over the gene-body positively correlated with degree of transcriptional activity. Importantly, CLL samples showed aberrant 5-hmC and 5-mC pattern over gene-body compared to well-defined patterns in normal B-cells. Integrated analysis of 5-hmC and RNA-sequencing from CLL datasets identified three novel oncogenic drivers that could have potential roles in CLL development and progression. CONCLUSIONS: Thus, our study suggests that the global loss of 5-hmC, accompanied by its significant increase at the gene regulatory regions, constitute a novel hallmark of CLL pathogenesis. Our combined analysis of 5-mC and 5-hmC sequencing provided insights into the potential role of 5-hmC in modulating gene expression changes during CLL pathogenesis.</p>',
'date' => '2019-01-07',
'pmid' => 'http://www.pubmed.gov/30616658',
'doi' => '10.1186/s13072‑018‑0252‑7',
'modified' => '2019-07-01 11:46:16',
'created' => '2019-06-21 14:55:31',
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(int) 3 => array(
'id' => '2992',
'name' => 'Regulation of the DNA Methylation Landscape in Human Somatic Cell Reprogramming by the miR-29 Family',
'authors' => 'Hysolli E et al.',
'description' => 'Reprogramming to pluripotency after overexpression of OCT4, SOX2, KLF4, and MYC is accompanied by global genomic and epigenomic changes. Histone modification and DNA methylation states in induced pluripotent stem cells (iPSCs) have been shown to be highly similar to embryonic stem cells (ESCs). However, epigenetic differences still exist between iPSCs and ESCs. In particular, aberrant DNA methylation states found in iPSCs are a major concern when using iPSCs in a clinical setting. Thus, it is critical to find factors that regulate DNA methylation states in reprogramming. Here, we found that the miR-29 family is an important epigenetic regulator during human somatic cell reprogramming. Our global DNA methylation and hydroxymethylation analysis shows that DNA demethylation is a major event mediated by miR-29a depletion during early reprogramming, and that iPSCs derived from miR-29a depletion are epigenetically closer to ESCs. Our findings uncover an important miRNA-based approach to generate clinically robust iPSCs.',
'date' => '2016-07-12',
'pmid' => 'http://www.ncbi.nlm.nih.gov/pubmed/27373925',
'doi' => '10.1016/j.stemcr.2016.05.014',
'modified' => '2016-08-23 09:57:29',
'created' => '2016-08-23 09:57:29',
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(int) 4 => array(
'id' => '2811',
'name' => 'Transcriptome-wide distribution and function of RNA hydroxymethylcytosine',
'authors' => 'Delatte B, Wang F, Ngoc LV, Collignon E, Bonvin E, Deplus R, Calonne E, Hassabi B, Putmans P, Awe S, Wetzel C, Kreher J, Soin R, Creppe C, Limbach PA, Gueydan C, Kruys V, Brehm A, Minakhina S, Defrance M, Steward R, Fuks F.',
'description' => '<p>Hydroxymethylcytosine, well described in DNA, occurs also in RNA. Here, we show that hydroxymethylcytosine preferentially marks polyadenylated RNAs and is deposited by Tet in Drosophila. We map the transcriptome-wide hydroxymethylation landscape, revealing hydroxymethylcytosine in the transcripts of many genes, notably in coding sequences, and identify consensus sites for hydroxymethylation. We found that RNA hydroxymethylation can favor mRNA translation. Tet and hydroxymethylated RNA are found to be most abundant in the Drosophila brain, and Tet-deficient fruitflies suffer impaired brain development, accompanied by decreased RNA hydroxymethylation. This study highlights the distribution, localization, and function of cytosine hydroxymethylation and identifies central roles for this modification in Drosophila.</p>',
'date' => '2016-01-15',
'pmid' => 'http://pubmed.gov/26816380',
'doi' => '10.1126/science.aac5253',
'modified' => '2016-01-28 21:57:55',
'created' => '2016-01-28 21:57:55',
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(int) 5 => array(
'id' => '3750',
'name' => 'RNA biochemistry. Transcriptome-wide distribution and function of RNA hydroxymethylcytosine.',
'authors' => 'Delatte B, Wang F, Ngoc LV, Collignon E, Bonvin E, Deplus R, Calonne E, Hassabi B, Putmans P, Awe S, Wetzel C, Kreher J, Soin R, Creppe C, Limbach PA, Gueydan C, Kruys V, Brehm A, Minakhina S, Defrance M, Steward R, Fuks F',
'description' => '<p>Hydroxymethylcytosine, well described in DNA, occurs also in RNA. Here, we show that hydroxymethylcytosine preferentially marks polyadenylated RNAs and is deposited by Tet in Drosophila. We map the transcriptome-wide hydroxymethylation landscape, revealing hydroxymethylcytosine in the transcripts of many genes, notably in coding sequences, and identify consensus sites for hydroxymethylation. We found that RNA hydroxymethylation can favor mRNA translation. Tet and hydroxymethylated RNA are found to be most abundant in the Drosophila brain, and Tet-deficient fruitflies suffer impaired brain development, accompanied by decreased RNA hydroxymethylation. This study highlights the distribution, localization, and function of cytosine hydroxymethylation and identifies central roles for this modification in Drosophila.</p>',
'date' => '2016-01-15',
'pmid' => 'http://www.pubmed.org/26816380',
'doi' => '10.1126/science.aac5253',
'modified' => '2019-10-03 12:29:05',
'created' => '2019-10-02 16:16:55',
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[maximum depth reached]
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(int) 6 => array(
'id' => '2613',
'name' => 'CpG signalling, H2A.Z/H3 acetylation and microRNA-mediated deferred self-attenuation orchestrate foetal NOS3 expression.',
'authors' => 'Postberg J, Kanders M, Forcob S, Willems R, Orth V, Hensel KO, Weil PP, Wirth S, Jenke AC',
'description' => 'BACKGROUND: An adverse intrauterine environment leads to permanent physiological changes including vascular tone regulation, potentially influencing the risk for adult vascular diseases. We therefore aimed to monitor responsive NOS3 expression in human umbilical artery endothelial cells (HUAEC) and to study the underlying epigenetic signatures involved in its regulation. RESULTS: NOS3 and STAT3 mRNA levels were elevated in HUAEC of patients who suffered from placental insufficiency. 5-hydroxymethylcytosine, H3K9ac and Histone 2A (H2A).Zac at the NOS3 transcription start site directly correlated with NOS3 mRNA levels. Concomitantly, we observed entangled histone acetylation patterns and NOS3 response upon hypoxic conditions in vitro. Knock-down of either NOS3 or STAT3 by RNAi provided evidence for a functional NOS3/STAT3 relationship. Moreover, we recognized massive turnover of Stat3 at a discrete binding site in the NOS3 promoter. Interestingly, induced hyperacetylation resulted in short-termed increase of NOS3 mRNA followed by deferred decrease indicating that NOS3 expression could become self-attenuated by co-expressed intronic 27 nt-ncRNA. Reporter assay results and phylogenetic analyses enabled us to propose a novel model for STAT3-3'-UTR targeting by this 27-nt-ncRNA. CONCLUSIONS: An adverse intrauterine environment leads to adaptive changes of NOS3 expression. Apparently, a rapid NOS3 self-limiting response upon ectopic triggers co-exists with longer termed expression changes in response to placental insufficiency involving differential epigenetic signatures. Their persistence might contribute to impaired vascular endothelial response and consequently increase the risk of cardiovascular disease later in life.',
'date' => '2015-01-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/25699114',
'doi' => '',
'modified' => '2015-07-24 15:39:05',
'created' => '2015-07-24 15:39:05',
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(int) 7 => array(
'id' => '2348',
'name' => 'White matter tract and glial-associated changes in 5-hydroxymethylcytosine following chronic cerebral hypoperfusion.',
'authors' => 'Tsenkina Y, Ruzov A, Gliddon C, Horsburgh K, De Sousa PA',
'description' => 'White matter abnormalities due to age-related cerebrovascular alterations is a common pathological hallmark associated with functional impairment in the elderly which has been modeled in chronically hypoperfused mice. 5-Methylcytosine (5mC) and its oxidized derivative 5-hydroxymethylcytosine (5hmC) are DNA modifications that have been recently linked with age-related neurodegeneration and cerebrovascular pathology. Here we conducted a pilot investigation of whether chronic cerebral hypoperfusion might affect genomic distribution of these modifications and/ or a Ten-Eleven Translocation protein 2 (TET2) which catalyses hydroxymethylation in white and grey matter regions of this animal model. Immunohistochemical evaluation of sham and chronically hypoperfused mice a month after surgery revealed significant (p<0.05) increases in the proportion of 5hmC positive cells, Iba1 positive inflammatory microglia, and NG2 positive oligodendroglial progenitors in the hypoperfused corpus callosum. In the same white matter tract there was an absence of hypoperfusion-induced alterations in the proportion of 5mC, TET2 positive cells and CC1 positive mature oligodrendrocytes. Correlation analysis across animals within both treatment groups demonstrated a significant association of the elevated 5hmC levels with increases in the proportion of inflammatory microglia only (p=0.01) in the corpus callosum. In vitro studies revealed that 5hmC is lost during oligodendroglial maturation but not microglial activation. Additionally, TET1, TET2, and TET3 protein levels showed dynamic alterations during oligodendroglial development and following oxidative stress in vitro. Our study suggests that 5hmC exhibits white matter tract and cell type specific dynamics following chronic cerebral hypoperfusion in mice.',
'date' => '2014-10-10',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/25305569',
'doi' => '',
'modified' => '2015-07-24 15:39:04',
'created' => '2015-07-24 15:39:04',
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),
(int) 8 => array(
'id' => '994',
'name' => 'Tet2 Facilitates the Derepression of Myeloid Target Genes during CEBPα-Induced Transdifferentiation of Pre-B Cells.',
'authors' => 'Kallin EM, Rodríguez-Ubreva J, Christensen J, Cimmino L, Aifantis I, Helin K, Ballestar E, Graf T',
'description' => 'The methylcytosine hydroxylase Tet2 has been implicated in hematopoietic differentiation and the formation of myeloid malignancies when mutated. An ideal system to study the role of Tet2 in myelopoeisis is CEBPα-induced transdifferentiation of pre-B cells into macrophages. Here we found that CEBPα binds to upstream regions of Tet2 and that the gene becomes activated. Tet2 knockdowns impaired the upregulation of macrophage markers as well as phagocytic capacity, suggesting that the enzyme is required for both early and late stage myeloid differentiation. A slightly weaker effect was seen in primary cells with a Tet2 ablation. Expression arrays of transdifferentiating cells with Tet2 knockdowns permitted the identification of a small subset of myeloid genes whose upregulation was blunted. Activation of these target genes was accompanied by rapid increases of promoter hydroxy-methylation. Our observations indicate that Tet2 helps CEBPα rapidly derepress myeloid genes during the conversion of pre-B cells into macrophages.',
'date' => '2012-09-12',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/22981865',
'doi' => '',
'modified' => '2015-07-24 15:38:59',
'created' => '2015-07-24 15:38:59',
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[maximum depth reached]
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(int) 9 => array(
'id' => '149',
'name' => 'Lineage-specific distribution of high levels of genomic 5-hydroxymethylcytosine in mammalian development.',
'authors' => 'Ruzov A, Tsenkina Y, Serio A, Dudnakova T, Fletcher J, Bai Y, Chebotareva T, Pells S, Hannoun Z, Sullivan G, Chandran S, Hay DC, Bradley M, Wilmut I, De Sousa P',
'description' => 'Methylation of cytosine is a DNA modification associated with gene repression. Recently, a novel cytosine modification, 5-hydroxymethylcytosine (5-hmC) has been discovered. Here we examine 5-hmC distribution during mammalian development and in cellular systems, and show that the developmental dynamics of 5-hmC are different from those of 5-methylcytosine (5-mC); in particular 5-hmC is enriched in embryonic contexts compared to adult tissues. A detectable 5-hmC signal appears in pre-implantation development starting at the zygote stage, where the paternal genome is subjected to a genome-wide hydroxylation of 5-mC, which precisely coincides with the loss of the 5-mC signal in the paternal pronucleus. Levels of 5-hmC are high in cells of the inner cell mass in blastocysts, and the modification colocalises with nestin-expressing cell populations in mouse post-implantation embryos. Compared to other adult mammalian organs, 5-hmC is strongly enriched in bone marrow and brain, wherein high 5-hmC content is a feature of both neuronal progenitors and post-mitotic neurons. We show that high levels of 5-hmC are not only present in mouse and human embryonic stem cells (ESCs) and lost during differentiation, as has been reported previously, but also reappear during the generation of induced pluripotent stem cells; thus 5-hmC enrichment correlates with a pluripotent cell state. Our findings suggest that apart from the cells of neuronal lineages, high levels of genomic 5-hmC are an epigenetic feature of embryonic cell populations and cellular pluri- and multi-lineage potency. To our knowledge, 5-hmC represents the first epigenetic modification of DNA discovered whose enrichment is so cell-type specific.',
'date' => '2011-09-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/21747414',
'doi' => '',
'modified' => '2015-07-24 15:38:57',
'created' => '2015-07-24 15:38:57',
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[maximum depth reached]
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(int) 10 => array(
'id' => '361',
'name' => 'Genome-wide analysis of 5-hydroxymethylcytosine distribution reveals its dual function in transcriptional regulation in mouse embryonic stem cells.',
'authors' => 'Wu H, D'Alessio AC, Ito S, Wang Z, Cui K, Zhao K, Sun YE, Zhang Y',
'description' => 'Recent studies have demonstrated that the Ten-eleven translocation (Tet) family proteins can enzymatically convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). While 5mC has been studied extensively, little is known about the distribution and function of 5hmC. Here we present a genome-wide profile of 5hmC in mouse embryonic stem (ES) cells. A combined analysis of global 5hmC distribution and gene expression profile in wild-type and Tet1-depleted ES cells suggests that 5hmC is enriched at both gene bodies of actively transcribed genes and extended promoter regions of Polycomb-repressed developmental regulators. Thus, our study reveals the first genome-wide 5hmC distribution in pluripotent stem cells, and supports its dual function in regulating gene expression.',
'date' => '2011-04-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/21460036',
'doi' => '',
'modified' => '2015-07-24 15:38:57',
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'id' => '2033',
'antibody_id' => '59',
'name' => '5-hydroxymethylcytosine (5-hmC) Antibody (rat) ',
'description' => '<p>5<span>-hmC is a DNA modification which results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. Preliminary results indicate that 5-hmC may have important roles distinct from 5-methylcytosine (5-mC). Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics.</span></p>',
'label1' => 'Validation Data',
'info1' => '<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig1.png" alt="hMeDIP" style="display: block; margin-left: auto; margin-right: auto;" /></p>
</div>
<div class="small-6 columns">
<p><small><strong>Figure 1. Hydroxymethylated DNA IP results obtained with our hMeDIP kit (Cat. No. AF-104-0016)</strong><br /> Hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit. This kit includes: the monoclonal antibody against 5-hydroxymethylcytosine (Cat. No. MAb-633HMC-050), 5-hmC, 5-mC & cytosine DNA standards & Rat IgG (Cat. No. AF-105-0025). The DNA was prepared with the GenDNA module and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. 1 μg of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP’d material has been analysed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control • with Rat IgG as negative control (0.06%, almost no recovery) • with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control • with Rat IgG as negative control (0.03%, almost no recovery) • with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control • with Rat IgG as negative control (0.04%, almost no recovery) • with 5-hmC (97.60% recovery, almost full recovery) These results clearly demonstrate the high specificity and efficiency of the 5-hydroxymethylcytosine monoclonal antibody.</small></p>
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</div>
<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig2.png" alt="ELISA" style="display: block; margin-left: auto; margin-right: auto;" width="375" height="274" /></p>
</div>
<div class="small-6 columns">
<p><small><strong>Figure 2. Determination of the 5-hmC rat monoclonal antibody titer</strong><br />To determine the titer, an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody directed against 5-hmC (Cat No. MAb-633HMC-050, MAb-633HMC-100) in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.</small></p>
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</div>
<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig3.png" alt="Dot blot" style="display: block; margin-left: auto; margin-right: auto;" width="190" height="192" /></p>
</div>
<div class="small-6 columns">
<p><small><strong>Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls</strong><br />Figure 3A: Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.</small></p>
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<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig4.png" style="display: block; margin-left: auto; margin-right: auto;" width="115" height="232" /></p>
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<div class="small-6 columns">
<p><small><strong>Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls</strong><br />200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode « 5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.</small></p>
</div>
</div>',
'label2' => 'Target description',
'info2' => '<p>5-hydroxymethylcytosine (5-hmC) has been recently discovered in mammalian DNA. This results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. So far, the 5-hmC bases have been identified in Purkinje neurons, in granule cells and embryonic stem cells where they are present at high levels (up to 0,6% of total nucleotides in Purkinje cells).</p>
<p>Preliminary results indicate that 5-hmC may have important roles distinct from 5-mC. Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics : 5-hydroxymethylcytosine may well represent a new pathway to demethylate DNA involving a repair mechanism converting 5-hmC to cytosine and, as such open up entirely new perspectives in epigenetic studies.</p>
<p>Due to the structural similarity between 5-mC and 5-hmC, these bases are experimentally almost indistinguishable. Recent articles demonstrated that the most common approaches (e.g. enzymatic approaches, bisulfite sequencing) do not account for 5-hmC. The development of the affinity-based technologies appears to be the most powerful way to differentially and specifically enrich 5-mC and 5-hmC sequences. The results shown here illustrate the use of this unique monoclonal antibody against 5-hydroxymethylcytosine that has been fully validated in various technologies.</p>',
'label3' => '',
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'format' => '50 µg',
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'type' => 'FRE',
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'slug' => '5-hmc-monoclonal-antibody-rat-classic-50-ug-32-ul',
'meta_title' => '5-hydroxymethylcytosine (5-hmC) Monoclonal Antibody (rat) | Diagenode',
'meta_keywords' => '5-hydroxymethylcytosine,5-hmC, 5-mC,monoclonal antibody ,Diagenode',
'meta_description' => '5-hydroxymethylcytosine (5-hmC) Monoclonal Antibody (rat) validated in hMeDIP, DB and ELISA. Batch-specific data available on the website. Sample size available',
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'id' => '2032',
'antibody_id' => '59',
'name' => '5-hydroxymethylcytosine (5-hmC) Antibody (rat) (sample size)',
'description' => '<p><span>5-hmC is a DNA modification which results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. Preliminary results indicate that 5-hmC may have important roles distinct from 5-methylcytosine (5-mC). Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics.</span></p>',
'label1' => 'Validation Data',
'info1' => '<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig1.png" alt="hMeDIP" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 1. Hydroxymethylated DNA IP results obtained with our hMeDIP kit (Cat. No. AF-104-0016)</strong><br /> Hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit. This kit includes: the monoclonal antibody against 5-hydroxymethylcytosine (Cat. No. MAb-633HMC-050), 5-hmC, 5-mC & cytosine DNA standards & Rat IgG (Cat. No. AF-105-0025). The DNA was prepared with the GenDNA module and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. 1 μg of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP’d material has been analysed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control • with Rat IgG as negative control (0.06%, almost no recovery) • with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control • with Rat IgG as negative control (0.03%, almost no recovery) • with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control • with Rat IgG as negative control (0.04%, almost no recovery) • with 5-hmC (97.60% recovery, almost full recovery) These results clearly demonstrate the high specificity and efficiency of the 5-hydroxymethylcytosine monoclonal antibody.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig2.png" alt="ELISA" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 2. Determination of the 5-hmC rat monoclonal antibody titer</strong><br /> To determine the titer, an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody directed against 5-hmC (Cat No. MAb-633HMC-050, MAb-633HMC-100) in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig3.png" alt="Dot blot" style="display: block; margin-left: auto; margin-right: auto;" width="200" height="200" /></p>
<p><strong>Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls</strong><br /> Figure 3A: Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig4.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls</strong><br /> 200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode « 5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.</p>',
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'name' => '5-hydroxymethylcytosine (5-hmC) Antibody (rat) ',
'description' => '<p>5<span>-hmC is a DNA modification which results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. Preliminary results indicate that 5-hmC may have important roles distinct from 5-methylcytosine (5-mC). Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics.</span></p>',
'label1' => 'Validation Data',
'info1' => '<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig1.png" alt="hMeDIP" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 1. Hydroxymethylated DNA IP results obtained with our hMeDIP kit (Cat. No. AF-104-0016)</strong><br /> Hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit. This kit includes: the monoclonal antibody against 5-hydroxymethylcytosine (Cat. No. MAb-633HMC-050), 5-hmC, 5-mC & cytosine DNA standards & Rat IgG (Cat. No. AF-105-0025). The DNA was prepared with the GenDNA module and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. 1 μg of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP’d material has been analysed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control • with Rat IgG as negative control (0.06%, almost no recovery) • with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control • with Rat IgG as negative control (0.03%, almost no recovery) • with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control • with Rat IgG as negative control (0.04%, almost no recovery) • with 5-hmC (97.60% recovery, almost full recovery) These results clearly demonstrate the high specificity and efficiency of the 5-hydroxymethylcytosine monoclonal antibody.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig2.png" alt="ELISA" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 2. Determination of the 5-hmC rat monoclonal antibody titer</strong><br /> To determine the titer, an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody directed against 5-hmC (Cat No. MAb-633HMC-050, MAb-633HMC-100) in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig3.png" alt="Dot blot" style="display: block; margin-left: auto; margin-right: auto;" width="200" height="200" /></p>
<p><strong>Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls</strong><br /> Figure 3A: Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig4.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls</strong><br /> 200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode « 5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.</p>',
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'format' => '100 µg',
'catalog_number' => 'C15220001-100',
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$related = array(
'id' => '1882',
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'name' => 'hMeDIP kit x16 (monoclonal mouse antibody)',
'description' => '<p><a href="https://www.diagenode.com/files/products/kits/hMeDIP_kit_manual.pdf"><img src="https://www.diagenode.com/img/buttons/bt-manual.png" /></a></p>
<p><span>The hMeDIP kit is designed for enrichment of hydroxymethylated DNA from fragmented genomic DNA<span><span> </span>samples for use in genome-wide methylation analysis. It features</span></span><span> a highly specific monoclonal antibody against </span>5-hydroxymethylcytosine (5-hmC) for the immunoprecipitation of hydroxymethylated DNA<span>. It includes control DNA and primers to assess the effiency of the assay. </span>Performing hydroxymethylation profiling with the hMeDIP kit is fast, reliable and highly specific.</p>
<p><em>Looking for hMeDIP-seq protocol? <a href="https://go.diagenode.com/l/928883/2022-01-07/2m1ht" target="_blank" title="Contact us">Contact us</a></em></p>
<p><span></span></p>
<p><span></span></p>',
'label1' => 'Characteristics',
'info1' => '<ul style="list-style-type: disc;">
<li><span>Robust enrichment & immunoprecipitation of hydroxymethylated DNA</span></li>
<li>Highly specific monoclonal antibody against 5-hmC<span> for reliable, reproducible results</span></li>
<li>Including control DNA and primers to <span>monitor the efficiency of the assay</span>
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<li>hmeDNA and unmethylated DNA sequences and primer pairs</li>
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<p><strong>Figure 2. Determination of the 5-hmC rat monoclonal antibody titer</strong><br /> To determine the titer, an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody directed against 5-hmC (Cat No. MAb-633HMC-050, MAb-633HMC-100) in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig3.png" alt="Dot blot" style="display: block; margin-left: auto; margin-right: auto;" width="200" height="200" /></p>
<p><strong>Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls</strong><br /> Figure 3A: Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig4.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls</strong><br /> 200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode « 5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.</p>',
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'description' => '<p>Cytosine hydroxymethylation was recently discovered as an important epigenetic mechanism. This cytosine base modification results from the enzymatic conversion of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC) by the TET family of oxygenases. Though the precise role of 5-hmC is the subject of intense research and debate, early studies strongly indicate that it is also involved in gene regulation and in numerous important biological processes including embryonic development, cellular differentiation, stem cell reprogramming and carcinogenesis.</p>
<p>The study of 5-hmC has long been limited due to the lack of high quality, validated tools and technologies that discriminate hydroxymethylation from methylation in regulating gene expression. The use of highly specific antibodies against 5-hmC for the immunoprecipitation of hydroxymethylated DNA offers a reliable solution for hydroxymethylation profiling.</p>
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'description' => 'Recent studies have demonstrated that the Ten-eleven translocation (Tet) family proteins can enzymatically convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). While 5mC has been studied extensively, little is known about the distribution and function of 5hmC. Here we present a genome-wide profile of 5hmC in mouse embryonic stem (ES) cells. A combined analysis of global 5hmC distribution and gene expression profile in wild-type and Tet1-depleted ES cells suggests that 5hmC is enriched at both gene bodies of actively transcribed genes and extended promoter regions of Polycomb-repressed developmental regulators. Thus, our study reveals the first genome-wide 5hmC distribution in pluripotent stem cells, and supports its dual function in regulating gene expression.',
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<p><strong>Figure 1. Hydroxymethylated DNA IP results obtained with our hMeDIP kit (Cat. No. AF-104-0016)</strong><br /> Hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit. This kit includes: the monoclonal antibody against 5-hydroxymethylcytosine (Cat. No. MAb-633HMC-050), 5-hmC, 5-mC & cytosine DNA standards & Rat IgG (Cat. No. AF-105-0025). The DNA was prepared with the GenDNA module and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. 1 μg of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP’d material has been analysed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control • with Rat IgG as negative control (0.06%, almost no recovery) • with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control • with Rat IgG as negative control (0.03%, almost no recovery) • with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control • with Rat IgG as negative control (0.04%, almost no recovery) • with 5-hmC (97.60% recovery, almost full recovery) These results clearly demonstrate the high specificity and efficiency of the 5-hydroxymethylcytosine monoclonal antibody.</p>
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<p><strong>Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls</strong><br /> Figure 3A: Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig4.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls</strong><br /> 200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode « 5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.</p>',
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'description' => '5-hydroxymethylcytosine (5-hmC) has been recently discovered in mammalian DNA. This results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. So far, the 5-hmC bases have been identified in Purkinje neurons, in granule cells and embryonic stem cells where they are present at high levels (up to 0,6% of total nucleotides in Purkinje cells).
Preliminary results indicate that 5-hmC may have important roles distinct from 5-mC. Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics : 5-hydroxymethylcytosine may well represent a new pathway to demethylate DNA involving a repair mechanism converting 5-hmC to cytosine and, as such open up entirely new perspectives in epigenetic studies.
Due to the structural similarity between 5-mC and 5-hmC, these bases are experimentally almost indistinguishable. Recent articles demonstrated that the most common approaches (e.g. enzymatic approaches, bisulfite sequencing) do not account for 5-hmC. The development of the affinity-based technologies appears to be the most powerful way to differentially and specifically enrich 5-mC and 5-hmC sequences. The results shown here illustrate the use of this unique monoclonal antibody against 5-hydroxymethylcytosine that has been fully validated in various technologies.',
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'description' => '<p>5<span>-hmC is a DNA modification which results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. Preliminary results indicate that 5-hmC may have important roles distinct from 5-methylcytosine (5-mC). Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics.</span></p>',
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'info1' => '<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig1.png" alt="hMeDIP" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 1. Hydroxymethylated DNA IP results obtained with our hMeDIP kit (Cat. No. AF-104-0016)</strong><br /> Hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit. This kit includes: the monoclonal antibody against 5-hydroxymethylcytosine (Cat. No. MAb-633HMC-050), 5-hmC, 5-mC & cytosine DNA standards & Rat IgG (Cat. No. AF-105-0025). The DNA was prepared with the GenDNA module and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. 1 μg of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP’d material has been analysed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control • with Rat IgG as negative control (0.06%, almost no recovery) • with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control • with Rat IgG as negative control (0.03%, almost no recovery) • with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control • with Rat IgG as negative control (0.04%, almost no recovery) • with 5-hmC (97.60% recovery, almost full recovery) These results clearly demonstrate the high specificity and efficiency of the 5-hydroxymethylcytosine monoclonal antibody.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig2.png" alt="ELISA" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 2. Determination of the 5-hmC rat monoclonal antibody titer</strong><br /> To determine the titer, an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody directed against 5-hmC (Cat No. MAb-633HMC-050, MAb-633HMC-100) in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig3.png" alt="Dot blot" style="display: block; margin-left: auto; margin-right: auto;" width="200" height="200" /></p>
<p><strong>Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls</strong><br /> Figure 3A: Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig4.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls</strong><br /> 200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode « 5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.</p>',
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<p>Preliminary results indicate that 5-hmC may have important roles distinct from 5-mC. Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics : 5-hydroxymethylcytosine may well represent a new pathway to demethylate DNA involving a repair mechanism converting 5-hmC to cytosine and, as such open up entirely new perspectives in epigenetic studies.</p>
<p>Due to the structural similarity between 5-mC and 5-hmC, these bases are experimentally almost indistinguishable. Recent articles demonstrated that the most common approaches (e.g. enzymatic approaches, bisulfite sequencing) do not account for 5-hmC. The development of the affinity-based technologies appears to be the most powerful way to differentially and specifically enrich 5-mC and 5-hmC sequences. The results shown here illustrate the use of this unique monoclonal antibody against 5-hydroxymethylcytosine that has been fully validated in various technologies.</p>',
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Preliminary results indicate that 5-hmC may have important roles distinct from 5-mC. Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics : 5-hydroxymethylcytosine may well represent a new pathway to demethylate DNA involving a repair mechanism converting 5-hmC to cytosine and, as such open up entirely new perspectives in epigenetic studies.
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<td>Fig 1</td>
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<td>Dot Blotting</td>
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'description' => '<p>5<span>-hmC is a DNA modification which results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. Preliminary results indicate that 5-hmC may have important roles distinct from 5-methylcytosine (5-mC). Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics.</span></p>',
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<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig1.png" alt="hMeDIP" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<p><small><strong>Figure 1. Hydroxymethylated DNA IP results obtained with our hMeDIP kit (Cat. No. AF-104-0016)</strong><br /> Hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit. This kit includes: the monoclonal antibody against 5-hydroxymethylcytosine (Cat. No. MAb-633HMC-050), 5-hmC, 5-mC & cytosine DNA standards & Rat IgG (Cat. No. AF-105-0025). The DNA was prepared with the GenDNA module and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. 1 μg of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP’d material has been analysed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control • with Rat IgG as negative control (0.06%, almost no recovery) • with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control • with Rat IgG as negative control (0.03%, almost no recovery) • with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control • with Rat IgG as negative control (0.04%, almost no recovery) • with 5-hmC (97.60% recovery, almost full recovery) These results clearly demonstrate the high specificity and efficiency of the 5-hydroxymethylcytosine monoclonal antibody.</small></p>
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<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig2.png" alt="ELISA" style="display: block; margin-left: auto; margin-right: auto;" width="375" height="274" /></p>
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<p><small><strong>Figure 2. Determination of the 5-hmC rat monoclonal antibody titer</strong><br />To determine the titer, an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody directed against 5-hmC (Cat No. MAb-633HMC-050, MAb-633HMC-100) in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.</small></p>
</div>
</div>
<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig3.png" alt="Dot blot" style="display: block; margin-left: auto; margin-right: auto;" width="190" height="192" /></p>
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<p><small><strong>Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls</strong><br />Figure 3A: Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.</small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig4.png" style="display: block; margin-left: auto; margin-right: auto;" width="115" height="232" /></p>
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<p><small><strong>Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls</strong><br />200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode « 5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.</small></p>
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<p>Preliminary results indicate that 5-hmC may have important roles distinct from 5-mC. Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics : 5-hydroxymethylcytosine may well represent a new pathway to demethylate DNA involving a repair mechanism converting 5-hmC to cytosine and, as such open up entirely new perspectives in epigenetic studies.</p>
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<h3>MBD-seq allows for detection of genomic regions with different CpG density</h3>
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<li><strong>High capture efficiency</strong></li>
<li><strong>Differential fractionation</strong> of methylated DNA by CpG density (3 eluted fractions)</li>
<li><strong>On-day protocol</strong></li>
<li><strong>NGS compatibility</strong></li>
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<h3>MBD-seq allows for detection of genomic regions with different CpG density</h3>
<p><img src="https://www.diagenode.com/img/product/kits/mbd_results1.png" alt="MBD-sequencing results have been validated by bisulfite sequencing" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong></strong></p>
<p><strong></strong><strong>F</strong><strong>igure 1.</strong> Using the MBD approach, two methylated regions were detected in different elution fractions according to their methylated CpG density (A). Low, Medium and High refer to the sequenced DNA from different elution fractions with increasing salt concentration. Methylated patterns of these two different methylated regions were validated by bisulfite conversion assay (B).</p>',
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<p style="text-align: center;"><strong>Make your Bisulfite conversion now in only 60 minutes !</strong></p>
<p>Diagenode's Premium Bisulfite Kit rapidly converts DNA through bisulfite treatment. Our conversion reagent is added directly to DNA, requires no intermediate steps, and results in high yields of DNA ready for downstream analysis methods including PCR and Next-Generation Sequencing.</p>',
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<p><span>The hMeDIP kit is designed for enrichment of hydroxymethylated DNA from fragmented genomic DNA<span><span> </span>samples for use in genome-wide methylation analysis. It features</span></span><span> a highly specific monoclonal antibody against </span>5-hydroxymethylcytosine (5-hmC) for the immunoprecipitation of hydroxymethylated DNA<span>. It includes control DNA and primers to assess the effiency of the assay. </span>Performing hydroxymethylation profiling with the hMeDIP kit is fast, reliable and highly specific.</p>
<p><em>Looking for hMeDIP-seq protocol? <a href="https://go.diagenode.com/l/928883/2022-01-07/2m1ht" target="_blank" title="Contact us">Contact us</a></em></p>
<p><span></span></p>
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<li>Including control DNA and primers to <span>monitor the efficiency of the assay</span>
<ul style="list-style-type: circle;">
<li>hmeDNA and unmethylated DNA sequences and primer pairs</li>
<li>Mouse primer pairs against Sfi1 targeting hydroxymethylated gene in mouse</li>
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<li>Improved single-tube, magnetic bead-based protocol</li>
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'description' => '<p><span style="font-weight: 400;">T</span><span style="font-weight: 400;">he pattern of <strong>DNA modifications</strong> is critical for genome stability and the control of gene expression in the cell. Methylation of 5-cytosine (5-mC), one of the best-studied epigenetic marks, is carried out by the <strong>DNA methyltransferases</strong> DNMT3A and B and DNMT1. DNMT3A and DNMT3B are responsible for </span><i><span style="font-weight: 400;">de novo</span></i><span style="font-weight: 400;"> DNA methylation, whereas DNMT1 maintains existing methylation. 5-mC undergoes active demethylation which is performed by the <strong>Ten-Eleven Translocation</strong> (TET) familly of DNA hydroxylases. The latter consists of 3 members TET1, 2 and 3. All 3 members catalyze the conversion of <strong>5-methylcytosine</strong> (5-mC) into <strong>5-hydroxymethylcytosine</strong> (5-hmC), and further into <strong>5-formylcytosine</strong> (5-fC) and <strong>5-carboxycytosine</strong> (5-caC). 5-fC and 5-caC can be converted to unmodified cytosine by <strong>Thymine DNA Glycosylase</strong> (TDG). It is not yet clear if 5-hmC, 5-fC and 5-caC have specific functions or are simply intermediates in the demethylation of 5-mC.</span></p>
<p><span style="font-weight: 400;">DNA methylation is generally considered as a repressive mark and is usually associated with gene silencing. It is essential that the balance between DNA methylation and demethylation is precisely maintained. Dysregulation of DNA methylation may lead to many different human diseases and is often observed in cancer cells.</span></p>
<p><span style="font-weight: 400;">Diagenode offers highly validated antibodies against different proteins involved in DNA modifications as well as against the modified bases allowing the study of all steps and intermediates in the DNA methylation/demethylation pathway:</span></p>
<p><img src="https://www.diagenode.com/img/categories/antibodies/dna-methylation.jpg" height="599" width="816" /></p>
<p><strong>Diagenode exclusively sources the original 5-methylcytosine monoclonal antibody (clone 33D3).</strong></p>
<p>Check out the list below to see all proposed antibodies for DNA modifications.</p>
<p>Diagenode’s highly validated antibodies:</p>
<ul>
<li>Highly sensitive and specific</li>
<li>Cost-effective (requires less antibody per reaction)</li>
<li>Batch-specific data is available on the website</li>
<li>Expert technical support</li>
<li>Sample sizes available</li>
<li>100% satisfaction guarantee</li>
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'description' => '<p><span style="font-weight: 400;">All Diagenode’s antibodies are listed below. Please, use our Quick search field to find the antibody of interest by target name, application, purity.</span></p>
<p><span style="font-weight: 400;">Diagenode’s highly validated antibodies:</span></p>
<ul>
<li>Highly sensitive and specific</li>
<li>Cost-effective (requires less antibody per reaction)</li>
<li>Batch-specific data is available on the website</li>
<li>Expert technical support</li>
<li>Sample sizes available</li>
<li>100% satisfaction guarantee</li>
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'description' => '<p>Cytosine hydroxymethylation was recently discovered as an important epigenetic mechanism. This cytosine base modification results from the enzymatic conversion of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC) by the TET family of oxygenases. Though the precise role of 5-hmC is the subject of intense research and debate, early studies strongly indicate that it is also involved in gene regulation and in numerous important biological processes including embryonic development, cellular differentiation, stem cell reprogramming and carcinogenesis.</p>
<p>The study of 5-hmC has long been limited due to the lack of high quality, validated tools and technologies that discriminate hydroxymethylation from methylation in regulating gene expression. The use of highly specific antibodies against 5-hmC for the immunoprecipitation of hydroxymethylated DNA offers a reliable solution for hydroxymethylation profiling.</p>
<p></p>',
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<p><span> </span></p>
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'name' => 'DNMT1 regulates the timing of DNA methylation by DNMT3 in anenzymatic activity-dependent manner in mouse embryonic stem cells.',
'authors' => 'Ito Takamasa et al.',
'description' => '<p>DNA methylation (DNAme; 5-methylcytosine, 5mC) plays an essential role in mammalian development, and the 5mC profile is regulated by a balance of opposing enzymatic activities: DNA methyltransferases (DNMTs) and Ten-eleven translocation dioxygenases (TETs). In mouse embryonic stem cells (ESCs), de novo DNAme by DNMT3 family enzymes, demethylation by the TET-mediated conversion of 5mC to 5-hydroxymethylation (5hmC), and maintenance of the remaining DNAme by DNMT1 are actively repeated throughout cell cycles, dynamically forming a constant 5mC profile. Nevertheless, the detailed mechanism and physiological significance of this active cyclic DNA modification in mouse ESCs remain unclear. Here by visualizing the localization of DNA modifications on metaphase chromosomes and comparing whole-genome methylation profiles before and after the mid-S phase in ESCs lacking Dnmt1 (1KO ESCs), we demonstrated that in 1KO ESCs, DNMT3-mediated remethylation was interrupted during and after DNA replication. This results in a marked asymmetry in the distribution of 5hmC between sister chromatids at mitosis, with one chromatid being almost no 5hmC. When introduced in 1KO ESCs, the catalytically inactive form of DNMT1 (DNMT1CI) induced an increase in DNAme in pericentric heterochromatin and the DNAme-independent repression of IAPEz, a retrotransposon family, in 1KO ESCs. However, DNMT1CI could not restore the ability of DNMT3 to methylate unmodified dsDNA de novo in S phase in 1KO ESCs. Furthermore, during in vitro differentiation into epiblasts, 1KO ESCs expressing DNMT1CI showed an even stronger tendency to differentiate into the primitive endoderm than 1KO ESCs and were readily reprogrammed into the primitive streak via an epiblast-like cell state, reconfirming the importance of DNMT1 enzymatic activity at the onset of epiblast differentiation. These results indicate a novel function of DNMT1, in which DNMT1 actively regulates the timing and genomic targets of de novo methylation by DNMT3 in an enzymatic activity-dependent and independent manner, respectively.</p>',
'date' => '2022-01-01',
'pmid' => 'https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0262277',
'doi' => '10.1371/journal.pone.0262277',
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'id' => '4045',
'name' => 'Functional role of Tet-mediated RNA hydroxymethylcytosine in mouse ES
cells and during differentiation.',
'authors' => 'Lan, Jie and Rajan, Nicholas and Bizet, Martin and Penning, Audrey and
Singh, Nitesh K and Guallar, Diana and Calonne, Emilie and Li Greci, Andrea
and Bonvin, Elise and Deplus, Rachel and Hsu, Phillip J and Nachtergaele,
Sigrid and Ma, Chengjie and Song, ',
'description' => 'Tet-enzyme-mediated 5-hydroxymethylation of cytosines in DNA plays a
crucial role in mouse embryonic stem cells (ESCs). In RNA also,
5-hydroxymethylcytosine (5hmC) has recently been evidenced, but its
physiological roles are still largely unknown. Here we show the
contribution and function of this mark in mouse ESCs and differentiating
embryoid bodies. Transcriptome-wide mapping in ESCs reveals hundreds of
messenger RNAs marked by 5hmC at sites characterized by a defined unique
consensus sequence and particular features. During differentiation a large
number of transcripts, including many encoding key pluripotency-related
factors (such as Eed and Jarid2), show decreased cytosine
hydroxymethylation. Using Tet-knockout ESCs, we find Tet enzymes to be
partly responsible for deposition of 5hmC in mRNA. A transcriptome-wide
search further reveals mRNA targets to which Tet1 and Tet2 bind, at sites
showing a topology similar to that of 5hmC sites. Tet-mediated RNA
hydroxymethylation is found to reduce the stability of crucial
pluripotency-promoting transcripts. We propose that RNA cytosine
5-hydroxymethylation by Tets is a mark of transcriptome flexibility,
inextricably linked to the balance between pluripotency and lineage
commitment.',
'date' => '2020-10-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/33009383',
'doi' => '10.1038/s41467-020-18729-6',
'modified' => '2021-02-18 10:21:53',
'created' => '2021-02-18 10:21:53',
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'id' => '3660',
'name' => 'Global distribution of DNA hydroxymethylation and DNA methylation in chronic lymphocytic leukemia.',
'authors' => 'Wernig-Zorc S, Yadav MP, Kopparapu PK, Bemark M, Kristjansdottir HL, Andersson PO, Kanduri C, Kanduri M',
'description' => '<p>BACKGROUND: Chronic lymphocytic leukemia (CLL) has been a good model system to understand the functional role of 5-methylcytosine (5-mC) in cancer progression. More recently, an oxidized form of 5-mC, 5-hydroxymethylcytosine (5-hmC) has gained lot of attention as a regulatory epigenetic modification with prognostic and diagnostic implications for several cancers. However, there is no global study exploring the role of 5-hydroxymethylcytosine (5-hmC) levels in CLL. Herein, using mass spectrometry and hMeDIP-sequencing, we analysed the dynamics of 5-hmC during B cell maturation and CLL pathogenesis. RESULTS: We show that naïve B-cells had higher levels of 5-hmC and 5-mC compared to non-class switched and class-switched memory B-cells. We found a significant decrease in global 5-mC levels in CLL patients (n = 15) compared to naïve and memory B cells, with no changes detected between the CLL prognostic groups. On the other hand, global 5-hmC levels of CLL patients were similar to memory B cells and reduced compared to naïve B cells. Interestingly, 5-hmC levels were increased at regulatory regions such as gene-body, CpG island shores and shelves and 5-hmC distribution over the gene-body positively correlated with degree of transcriptional activity. Importantly, CLL samples showed aberrant 5-hmC and 5-mC pattern over gene-body compared to well-defined patterns in normal B-cells. Integrated analysis of 5-hmC and RNA-sequencing from CLL datasets identified three novel oncogenic drivers that could have potential roles in CLL development and progression. CONCLUSIONS: Thus, our study suggests that the global loss of 5-hmC, accompanied by its significant increase at the gene regulatory regions, constitute a novel hallmark of CLL pathogenesis. Our combined analysis of 5-mC and 5-hmC sequencing provided insights into the potential role of 5-hmC in modulating gene expression changes during CLL pathogenesis.</p>',
'date' => '2019-01-07',
'pmid' => 'http://www.pubmed.gov/30616658',
'doi' => '10.1186/s13072‑018‑0252‑7',
'modified' => '2019-07-01 11:46:16',
'created' => '2019-06-21 14:55:31',
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(int) 3 => array(
'id' => '2992',
'name' => 'Regulation of the DNA Methylation Landscape in Human Somatic Cell Reprogramming by the miR-29 Family',
'authors' => 'Hysolli E et al.',
'description' => 'Reprogramming to pluripotency after overexpression of OCT4, SOX2, KLF4, and MYC is accompanied by global genomic and epigenomic changes. Histone modification and DNA methylation states in induced pluripotent stem cells (iPSCs) have been shown to be highly similar to embryonic stem cells (ESCs). However, epigenetic differences still exist between iPSCs and ESCs. In particular, aberrant DNA methylation states found in iPSCs are a major concern when using iPSCs in a clinical setting. Thus, it is critical to find factors that regulate DNA methylation states in reprogramming. Here, we found that the miR-29 family is an important epigenetic regulator during human somatic cell reprogramming. Our global DNA methylation and hydroxymethylation analysis shows that DNA demethylation is a major event mediated by miR-29a depletion during early reprogramming, and that iPSCs derived from miR-29a depletion are epigenetically closer to ESCs. Our findings uncover an important miRNA-based approach to generate clinically robust iPSCs.',
'date' => '2016-07-12',
'pmid' => 'http://www.ncbi.nlm.nih.gov/pubmed/27373925',
'doi' => '10.1016/j.stemcr.2016.05.014',
'modified' => '2016-08-23 09:57:29',
'created' => '2016-08-23 09:57:29',
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(int) 4 => array(
'id' => '2811',
'name' => 'Transcriptome-wide distribution and function of RNA hydroxymethylcytosine',
'authors' => 'Delatte B, Wang F, Ngoc LV, Collignon E, Bonvin E, Deplus R, Calonne E, Hassabi B, Putmans P, Awe S, Wetzel C, Kreher J, Soin R, Creppe C, Limbach PA, Gueydan C, Kruys V, Brehm A, Minakhina S, Defrance M, Steward R, Fuks F.',
'description' => '<p>Hydroxymethylcytosine, well described in DNA, occurs also in RNA. Here, we show that hydroxymethylcytosine preferentially marks polyadenylated RNAs and is deposited by Tet in Drosophila. We map the transcriptome-wide hydroxymethylation landscape, revealing hydroxymethylcytosine in the transcripts of many genes, notably in coding sequences, and identify consensus sites for hydroxymethylation. We found that RNA hydroxymethylation can favor mRNA translation. Tet and hydroxymethylated RNA are found to be most abundant in the Drosophila brain, and Tet-deficient fruitflies suffer impaired brain development, accompanied by decreased RNA hydroxymethylation. This study highlights the distribution, localization, and function of cytosine hydroxymethylation and identifies central roles for this modification in Drosophila.</p>',
'date' => '2016-01-15',
'pmid' => 'http://pubmed.gov/26816380',
'doi' => '10.1126/science.aac5253',
'modified' => '2016-01-28 21:57:55',
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'id' => '3750',
'name' => 'RNA biochemistry. Transcriptome-wide distribution and function of RNA hydroxymethylcytosine.',
'authors' => 'Delatte B, Wang F, Ngoc LV, Collignon E, Bonvin E, Deplus R, Calonne E, Hassabi B, Putmans P, Awe S, Wetzel C, Kreher J, Soin R, Creppe C, Limbach PA, Gueydan C, Kruys V, Brehm A, Minakhina S, Defrance M, Steward R, Fuks F',
'description' => '<p>Hydroxymethylcytosine, well described in DNA, occurs also in RNA. Here, we show that hydroxymethylcytosine preferentially marks polyadenylated RNAs and is deposited by Tet in Drosophila. We map the transcriptome-wide hydroxymethylation landscape, revealing hydroxymethylcytosine in the transcripts of many genes, notably in coding sequences, and identify consensus sites for hydroxymethylation. We found that RNA hydroxymethylation can favor mRNA translation. Tet and hydroxymethylated RNA are found to be most abundant in the Drosophila brain, and Tet-deficient fruitflies suffer impaired brain development, accompanied by decreased RNA hydroxymethylation. This study highlights the distribution, localization, and function of cytosine hydroxymethylation and identifies central roles for this modification in Drosophila.</p>',
'date' => '2016-01-15',
'pmid' => 'http://www.pubmed.org/26816380',
'doi' => '10.1126/science.aac5253',
'modified' => '2019-10-03 12:29:05',
'created' => '2019-10-02 16:16:55',
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'id' => '2613',
'name' => 'CpG signalling, H2A.Z/H3 acetylation and microRNA-mediated deferred self-attenuation orchestrate foetal NOS3 expression.',
'authors' => 'Postberg J, Kanders M, Forcob S, Willems R, Orth V, Hensel KO, Weil PP, Wirth S, Jenke AC',
'description' => 'BACKGROUND: An adverse intrauterine environment leads to permanent physiological changes including vascular tone regulation, potentially influencing the risk for adult vascular diseases. We therefore aimed to monitor responsive NOS3 expression in human umbilical artery endothelial cells (HUAEC) and to study the underlying epigenetic signatures involved in its regulation. RESULTS: NOS3 and STAT3 mRNA levels were elevated in HUAEC of patients who suffered from placental insufficiency. 5-hydroxymethylcytosine, H3K9ac and Histone 2A (H2A).Zac at the NOS3 transcription start site directly correlated with NOS3 mRNA levels. Concomitantly, we observed entangled histone acetylation patterns and NOS3 response upon hypoxic conditions in vitro. Knock-down of either NOS3 or STAT3 by RNAi provided evidence for a functional NOS3/STAT3 relationship. Moreover, we recognized massive turnover of Stat3 at a discrete binding site in the NOS3 promoter. Interestingly, induced hyperacetylation resulted in short-termed increase of NOS3 mRNA followed by deferred decrease indicating that NOS3 expression could become self-attenuated by co-expressed intronic 27 nt-ncRNA. Reporter assay results and phylogenetic analyses enabled us to propose a novel model for STAT3-3'-UTR targeting by this 27-nt-ncRNA. CONCLUSIONS: An adverse intrauterine environment leads to adaptive changes of NOS3 expression. Apparently, a rapid NOS3 self-limiting response upon ectopic triggers co-exists with longer termed expression changes in response to placental insufficiency involving differential epigenetic signatures. Their persistence might contribute to impaired vascular endothelial response and consequently increase the risk of cardiovascular disease later in life.',
'date' => '2015-01-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/25699114',
'doi' => '',
'modified' => '2015-07-24 15:39:05',
'created' => '2015-07-24 15:39:05',
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'id' => '2348',
'name' => 'White matter tract and glial-associated changes in 5-hydroxymethylcytosine following chronic cerebral hypoperfusion.',
'authors' => 'Tsenkina Y, Ruzov A, Gliddon C, Horsburgh K, De Sousa PA',
'description' => 'White matter abnormalities due to age-related cerebrovascular alterations is a common pathological hallmark associated with functional impairment in the elderly which has been modeled in chronically hypoperfused mice. 5-Methylcytosine (5mC) and its oxidized derivative 5-hydroxymethylcytosine (5hmC) are DNA modifications that have been recently linked with age-related neurodegeneration and cerebrovascular pathology. Here we conducted a pilot investigation of whether chronic cerebral hypoperfusion might affect genomic distribution of these modifications and/ or a Ten-Eleven Translocation protein 2 (TET2) which catalyses hydroxymethylation in white and grey matter regions of this animal model. Immunohistochemical evaluation of sham and chronically hypoperfused mice a month after surgery revealed significant (p<0.05) increases in the proportion of 5hmC positive cells, Iba1 positive inflammatory microglia, and NG2 positive oligodendroglial progenitors in the hypoperfused corpus callosum. In the same white matter tract there was an absence of hypoperfusion-induced alterations in the proportion of 5mC, TET2 positive cells and CC1 positive mature oligodrendrocytes. Correlation analysis across animals within both treatment groups demonstrated a significant association of the elevated 5hmC levels with increases in the proportion of inflammatory microglia only (p=0.01) in the corpus callosum. In vitro studies revealed that 5hmC is lost during oligodendroglial maturation but not microglial activation. Additionally, TET1, TET2, and TET3 protein levels showed dynamic alterations during oligodendroglial development and following oxidative stress in vitro. Our study suggests that 5hmC exhibits white matter tract and cell type specific dynamics following chronic cerebral hypoperfusion in mice.',
'date' => '2014-10-10',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/25305569',
'doi' => '',
'modified' => '2015-07-24 15:39:04',
'created' => '2015-07-24 15:39:04',
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'id' => '994',
'name' => 'Tet2 Facilitates the Derepression of Myeloid Target Genes during CEBPα-Induced Transdifferentiation of Pre-B Cells.',
'authors' => 'Kallin EM, Rodríguez-Ubreva J, Christensen J, Cimmino L, Aifantis I, Helin K, Ballestar E, Graf T',
'description' => 'The methylcytosine hydroxylase Tet2 has been implicated in hematopoietic differentiation and the formation of myeloid malignancies when mutated. An ideal system to study the role of Tet2 in myelopoeisis is CEBPα-induced transdifferentiation of pre-B cells into macrophages. Here we found that CEBPα binds to upstream regions of Tet2 and that the gene becomes activated. Tet2 knockdowns impaired the upregulation of macrophage markers as well as phagocytic capacity, suggesting that the enzyme is required for both early and late stage myeloid differentiation. A slightly weaker effect was seen in primary cells with a Tet2 ablation. Expression arrays of transdifferentiating cells with Tet2 knockdowns permitted the identification of a small subset of myeloid genes whose upregulation was blunted. Activation of these target genes was accompanied by rapid increases of promoter hydroxy-methylation. Our observations indicate that Tet2 helps CEBPα rapidly derepress myeloid genes during the conversion of pre-B cells into macrophages.',
'date' => '2012-09-12',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/22981865',
'doi' => '',
'modified' => '2015-07-24 15:38:59',
'created' => '2015-07-24 15:38:59',
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(int) 9 => array(
'id' => '149',
'name' => 'Lineage-specific distribution of high levels of genomic 5-hydroxymethylcytosine in mammalian development.',
'authors' => 'Ruzov A, Tsenkina Y, Serio A, Dudnakova T, Fletcher J, Bai Y, Chebotareva T, Pells S, Hannoun Z, Sullivan G, Chandran S, Hay DC, Bradley M, Wilmut I, De Sousa P',
'description' => 'Methylation of cytosine is a DNA modification associated with gene repression. Recently, a novel cytosine modification, 5-hydroxymethylcytosine (5-hmC) has been discovered. Here we examine 5-hmC distribution during mammalian development and in cellular systems, and show that the developmental dynamics of 5-hmC are different from those of 5-methylcytosine (5-mC); in particular 5-hmC is enriched in embryonic contexts compared to adult tissues. A detectable 5-hmC signal appears in pre-implantation development starting at the zygote stage, where the paternal genome is subjected to a genome-wide hydroxylation of 5-mC, which precisely coincides with the loss of the 5-mC signal in the paternal pronucleus. Levels of 5-hmC are high in cells of the inner cell mass in blastocysts, and the modification colocalises with nestin-expressing cell populations in mouse post-implantation embryos. Compared to other adult mammalian organs, 5-hmC is strongly enriched in bone marrow and brain, wherein high 5-hmC content is a feature of both neuronal progenitors and post-mitotic neurons. We show that high levels of 5-hmC are not only present in mouse and human embryonic stem cells (ESCs) and lost during differentiation, as has been reported previously, but also reappear during the generation of induced pluripotent stem cells; thus 5-hmC enrichment correlates with a pluripotent cell state. Our findings suggest that apart from the cells of neuronal lineages, high levels of genomic 5-hmC are an epigenetic feature of embryonic cell populations and cellular pluri- and multi-lineage potency. To our knowledge, 5-hmC represents the first epigenetic modification of DNA discovered whose enrichment is so cell-type specific.',
'date' => '2011-09-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/21747414',
'doi' => '',
'modified' => '2015-07-24 15:38:57',
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'id' => '361',
'name' => 'Genome-wide analysis of 5-hydroxymethylcytosine distribution reveals its dual function in transcriptional regulation in mouse embryonic stem cells.',
'authors' => 'Wu H, D'Alessio AC, Ito S, Wang Z, Cui K, Zhao K, Sun YE, Zhang Y',
'description' => 'Recent studies have demonstrated that the Ten-eleven translocation (Tet) family proteins can enzymatically convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). While 5mC has been studied extensively, little is known about the distribution and function of 5hmC. Here we present a genome-wide profile of 5hmC in mouse embryonic stem (ES) cells. A combined analysis of global 5hmC distribution and gene expression profile in wild-type and Tet1-depleted ES cells suggests that 5hmC is enriched at both gene bodies of actively transcribed genes and extended promoter regions of Polycomb-repressed developmental regulators. Thus, our study reveals the first genome-wide 5hmC distribution in pluripotent stem cells, and supports its dual function in regulating gene expression.',
'date' => '2011-04-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/21460036',
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'id' => '2033',
'antibody_id' => '59',
'name' => '5-hydroxymethylcytosine (5-hmC) Antibody (rat) ',
'description' => '<p>5<span>-hmC is a DNA modification which results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. Preliminary results indicate that 5-hmC may have important roles distinct from 5-methylcytosine (5-mC). Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics.</span></p>',
'label1' => 'Validation Data',
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<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig1.png" alt="hMeDIP" style="display: block; margin-left: auto; margin-right: auto;" /></p>
</div>
<div class="small-6 columns">
<p><small><strong>Figure 1. Hydroxymethylated DNA IP results obtained with our hMeDIP kit (Cat. No. AF-104-0016)</strong><br /> Hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit. This kit includes: the monoclonal antibody against 5-hydroxymethylcytosine (Cat. No. MAb-633HMC-050), 5-hmC, 5-mC & cytosine DNA standards & Rat IgG (Cat. No. AF-105-0025). The DNA was prepared with the GenDNA module and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. 1 μg of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP’d material has been analysed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control • with Rat IgG as negative control (0.06%, almost no recovery) • with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control • with Rat IgG as negative control (0.03%, almost no recovery) • with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control • with Rat IgG as negative control (0.04%, almost no recovery) • with 5-hmC (97.60% recovery, almost full recovery) These results clearly demonstrate the high specificity and efficiency of the 5-hydroxymethylcytosine monoclonal antibody.</small></p>
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<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig2.png" alt="ELISA" style="display: block; margin-left: auto; margin-right: auto;" width="375" height="274" /></p>
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<div class="small-6 columns">
<p><small><strong>Figure 2. Determination of the 5-hmC rat monoclonal antibody titer</strong><br />To determine the titer, an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody directed against 5-hmC (Cat No. MAb-633HMC-050, MAb-633HMC-100) in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.</small></p>
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</div>
<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig3.png" alt="Dot blot" style="display: block; margin-left: auto; margin-right: auto;" width="190" height="192" /></p>
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<div class="small-6 columns">
<p><small><strong>Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls</strong><br />Figure 3A: Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.</small></p>
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<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig4.png" style="display: block; margin-left: auto; margin-right: auto;" width="115" height="232" /></p>
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<div class="small-6 columns">
<p><small><strong>Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls</strong><br />200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode « 5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.</small></p>
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</div>',
'label2' => 'Target description',
'info2' => '<p>5-hydroxymethylcytosine (5-hmC) has been recently discovered in mammalian DNA. This results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. So far, the 5-hmC bases have been identified in Purkinje neurons, in granule cells and embryonic stem cells where they are present at high levels (up to 0,6% of total nucleotides in Purkinje cells).</p>
<p>Preliminary results indicate that 5-hmC may have important roles distinct from 5-mC. Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics : 5-hydroxymethylcytosine may well represent a new pathway to demethylate DNA involving a repair mechanism converting 5-hmC to cytosine and, as such open up entirely new perspectives in epigenetic studies.</p>
<p>Due to the structural similarity between 5-mC and 5-hmC, these bases are experimentally almost indistinguishable. Recent articles demonstrated that the most common approaches (e.g. enzymatic approaches, bisulfite sequencing) do not account for 5-hmC. The development of the affinity-based technologies appears to be the most powerful way to differentially and specifically enrich 5-mC and 5-hmC sequences. The results shown here illustrate the use of this unique monoclonal antibody against 5-hydroxymethylcytosine that has been fully validated in various technologies.</p>',
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'meta_keywords' => '5-hydroxymethylcytosine,5-hmC, 5-mC,monoclonal antibody ,Diagenode',
'meta_description' => '5-hydroxymethylcytosine (5-hmC) Monoclonal Antibody (rat) validated in hMeDIP, DB and ELISA. Batch-specific data available on the website. Sample size available',
'modified' => '2024-11-19 16:58:50',
'created' => '2015-06-29 14:08:20'
),
(int) 1 => array(
'id' => '2032',
'antibody_id' => '59',
'name' => '5-hydroxymethylcytosine (5-hmC) Antibody (rat) (sample size)',
'description' => '<p><span>5-hmC is a DNA modification which results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. Preliminary results indicate that 5-hmC may have important roles distinct from 5-methylcytosine (5-mC). Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics.</span></p>',
'label1' => 'Validation Data',
'info1' => '<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig1.png" alt="hMeDIP" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 1. Hydroxymethylated DNA IP results obtained with our hMeDIP kit (Cat. No. AF-104-0016)</strong><br /> Hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit. This kit includes: the monoclonal antibody against 5-hydroxymethylcytosine (Cat. No. MAb-633HMC-050), 5-hmC, 5-mC & cytosine DNA standards & Rat IgG (Cat. No. AF-105-0025). The DNA was prepared with the GenDNA module and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. 1 μg of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP’d material has been analysed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control • with Rat IgG as negative control (0.06%, almost no recovery) • with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control • with Rat IgG as negative control (0.03%, almost no recovery) • with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control • with Rat IgG as negative control (0.04%, almost no recovery) • with 5-hmC (97.60% recovery, almost full recovery) These results clearly demonstrate the high specificity and efficiency of the 5-hydroxymethylcytosine monoclonal antibody.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig2.png" alt="ELISA" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 2. Determination of the 5-hmC rat monoclonal antibody titer</strong><br /> To determine the titer, an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody directed against 5-hmC (Cat No. MAb-633HMC-050, MAb-633HMC-100) in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig3.png" alt="Dot blot" style="display: block; margin-left: auto; margin-right: auto;" width="200" height="200" /></p>
<p><strong>Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls</strong><br /> Figure 3A: Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig4.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls</strong><br /> 200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode « 5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.</p>',
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'description' => '<p>5<span>-hmC is a DNA modification which results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. Preliminary results indicate that 5-hmC may have important roles distinct from 5-methylcytosine (5-mC). Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics.</span></p>',
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<p><strong>Figure 1. Hydroxymethylated DNA IP results obtained with our hMeDIP kit (Cat. No. AF-104-0016)</strong><br /> Hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit. This kit includes: the monoclonal antibody against 5-hydroxymethylcytosine (Cat. No. MAb-633HMC-050), 5-hmC, 5-mC & cytosine DNA standards & Rat IgG (Cat. No. AF-105-0025). The DNA was prepared with the GenDNA module and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. 1 μg of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP’d material has been analysed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control • with Rat IgG as negative control (0.06%, almost no recovery) • with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control • with Rat IgG as negative control (0.03%, almost no recovery) • with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control • with Rat IgG as negative control (0.04%, almost no recovery) • with 5-hmC (97.60% recovery, almost full recovery) These results clearly demonstrate the high specificity and efficiency of the 5-hydroxymethylcytosine monoclonal antibody.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig2.png" alt="ELISA" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 2. Determination of the 5-hmC rat monoclonal antibody titer</strong><br /> To determine the titer, an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody directed against 5-hmC (Cat No. MAb-633HMC-050, MAb-633HMC-100) in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig3.png" alt="Dot blot" style="display: block; margin-left: auto; margin-right: auto;" width="200" height="200" /></p>
<p><strong>Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls</strong><br /> Figure 3A: Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig4.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls</strong><br /> 200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode « 5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.</p>',
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<p><span>The hMeDIP kit is designed for enrichment of hydroxymethylated DNA from fragmented genomic DNA<span><span> </span>samples for use in genome-wide methylation analysis. It features</span></span><span> a highly specific monoclonal antibody against </span>5-hydroxymethylcytosine (5-hmC) for the immunoprecipitation of hydroxymethylated DNA<span>. It includes control DNA and primers to assess the effiency of the assay. </span>Performing hydroxymethylation profiling with the hMeDIP kit is fast, reliable and highly specific.</p>
<p><em>Looking for hMeDIP-seq protocol? <a href="https://go.diagenode.com/l/928883/2022-01-07/2m1ht" target="_blank" title="Contact us">Contact us</a></em></p>
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'name' => '5-hydroxymethylcytosine (5-hmC) Antibody (rat) (sample size)',
'description' => '<p><span>5-hmC is a DNA modification which results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. Preliminary results indicate that 5-hmC may have important roles distinct from 5-methylcytosine (5-mC). Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics.</span></p>',
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'info1' => '<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig1.png" alt="hMeDIP" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 1. Hydroxymethylated DNA IP results obtained with our hMeDIP kit (Cat. No. AF-104-0016)</strong><br /> Hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit. This kit includes: the monoclonal antibody against 5-hydroxymethylcytosine (Cat. No. MAb-633HMC-050), 5-hmC, 5-mC & cytosine DNA standards & Rat IgG (Cat. No. AF-105-0025). The DNA was prepared with the GenDNA module and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. 1 μg of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP’d material has been analysed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control • with Rat IgG as negative control (0.06%, almost no recovery) • with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control • with Rat IgG as negative control (0.03%, almost no recovery) • with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control • with Rat IgG as negative control (0.04%, almost no recovery) • with 5-hmC (97.60% recovery, almost full recovery) These results clearly demonstrate the high specificity and efficiency of the 5-hydroxymethylcytosine monoclonal antibody.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig2.png" alt="ELISA" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 2. Determination of the 5-hmC rat monoclonal antibody titer</strong><br /> To determine the titer, an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody directed against 5-hmC (Cat No. MAb-633HMC-050, MAb-633HMC-100) in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig3.png" alt="Dot blot" style="display: block; margin-left: auto; margin-right: auto;" width="200" height="200" /></p>
<p><strong>Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls</strong><br /> Figure 3A: Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15220001-fig4.png" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<p><strong>Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls</strong><br /> 200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode « 5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.</p>',
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'format' => '20 µg',
'catalog_number' => 'C15220001-20',
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'sf_code' => 'C15220001-D001-000831',
'type' => 'FRE',
'search_order' => '03-Antibody',
'price_EUR' => '125',
'price_USD' => '125',
'price_GBP' => '115',
'price_JPY' => '19580',
'price_CNY' => '',
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'meta_title' => '5-hmC Monoclonal Antibody (rat) | Diagenode',
'meta_keywords' => '',
'meta_description' => '5-hydroxymethylcytosine (5-hmC) Monoclonal Antibody (rat) validated in hMeDIP, DB and ELISA. Batch-specific data available on the website. Sample size available.',
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'name' => 'Dot blot protocol for 5-hydroxymethylcytosine monoclonal rat antibody',
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<p><span> </span></p>
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'type' => 'Protocol',
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'slug' => 'protocol-dot-blot5-hmc',
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'modified' => '2015-10-01 19:00:42',
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'meta_description' => 'Diagenode offers Monoclonal & Polyclonal antibodies for ELISA applications',
'meta_title' => 'ELISA Antibodies - Monoclonal & Polyclonal antibody | Diagenode',
'modified' => '2016-01-13 12:21:41',
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'description' => '<div class="row">
<div class="small-12 medium-12 large-12 columns">Enzyme-linked immunosorbent assay.</div>
</div>',
'in_footer' => false,
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'online' => true,
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'slug' => 'elisa-antibodies',
'meta_keywords' => ' ELISA Antibodies,Monoclonal antibody, Polyclonal antibody',
'meta_description' => 'Diagenode offers Monoclonal & Polyclonal antibodies for ELISA applications',
'meta_title' => 'ELISA Antibodies - Monoclonal & Polyclonal antibody | Diagenode',
'modified' => '2016-01-13 12:21:41',
'created' => '2014-07-08 08:13:28',
'locale' => 'eng'
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$description = '<div class="row">
<div class="small-12 medium-12 large-12 columns">Enzyme-linked immunosorbent assay.</div>
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'id' => '5',
'name' => 'Exclusive Highly Specific Kits Antibodies for DNA HydroxyMethylation Studies',
'description' => '<p>Cytosine hydroxymethylation was recently discovered as an important epigenetic mechanism. This cytosine base modification results from the enzymatic conversion of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC) by the TET family of oxygenases. Though the precise role of 5-hmC is the subject of intense research and debate, early studies strongly indicate that it is also involved in gene regulation and in numerous important biological processes including embryonic development, cellular differentiation, stem cell reprogramming and carcinogenesis.</p>
<p>The study of 5-hmC has long been limited due to the lack of high quality, validated tools and technologies that discriminate hydroxymethylation from methylation in regulating gene expression. The use of highly specific antibodies against 5-hmC for the immunoprecipitation of hydroxymethylated DNA offers a reliable solution for hydroxymethylation profiling.</p>
<p></p>',
'image_id' => null,
'type' => 'Poster',
'url' => 'files/posters/Exclusive_Highly_Specific_Kits_Antibodies_for_DNA_HydroxyMethylation_Studies_Poster.pdf',
'slug' => 'exclusive-highly-specific-kits-antibodies-for-dna-hydroxymethylation-studies-poster',
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'modified' => '2020-11-23 17:39:14',
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'id' => '484',
'name' => '5-hmC antibody rat SDS ES es',
'language' => 'es',
'url' => 'files/SDS/5-hmC/SDS-C15220001-5-hydroxymethylcytosine_5-hmC_Antibody_rat_-ES-es-GHS_2_0.pdf',
'countries' => 'ES',
'modified' => '2020-07-01 11:28:03',
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$publication = array(
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'name' => 'Genome-wide analysis of 5-hydroxymethylcytosine distribution reveals its dual function in transcriptional regulation in mouse embryonic stem cells.',
'authors' => 'Wu H, D'Alessio AC, Ito S, Wang Z, Cui K, Zhao K, Sun YE, Zhang Y',
'description' => 'Recent studies have demonstrated that the Ten-eleven translocation (Tet) family proteins can enzymatically convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). While 5mC has been studied extensively, little is known about the distribution and function of 5hmC. Here we present a genome-wide profile of 5hmC in mouse embryonic stem (ES) cells. A combined analysis of global 5hmC distribution and gene expression profile in wild-type and Tet1-depleted ES cells suggests that 5hmC is enriched at both gene bodies of actively transcribed genes and extended promoter regions of Polycomb-repressed developmental regulators. Thus, our study reveals the first genome-wide 5hmC distribution in pluripotent stem cells, and supports its dual function in regulating gene expression.',
'date' => '2011-04-01',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/21460036',
'doi' => '',
'modified' => '2015-07-24 15:38:57',
'created' => '2015-07-24 15:38:57',
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$externalLink = ' <a href="https://www.ncbi.nlm.nih.gov/pubmed/21460036" target="_blank"><i class="fa fa-external-link"></i></a>'include - APP/View/Products/view.ctp, line 755
View::_evaluate() - CORE/Cake/View/View.php, line 971
View::_render() - CORE/Cake/View/View.php, line 933
View::render() - CORE/Cake/View/View.php, line 473
Controller::render() - CORE/Cake/Controller/Controller.php, line 963
ProductsController::slug() - APP/Controller/ProductsController.php, line 1052
ReflectionMethod::invokeArgs() - [internal], line ??
Controller::invokeAction() - CORE/Cake/Controller/Controller.php, line 491
Dispatcher::_invoke() - CORE/Cake/Routing/Dispatcher.php, line 193
Dispatcher::dispatch() - CORE/Cake/Routing/Dispatcher.php, line 167
[main] - APP/webroot/index.php, line 118
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