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'description' => '<p><span>Polyclonal antibody raised in rabbit against the region of histone H4 containing the trimethylated lysine 20 (H4K20me3), using a KLH-conjugated synthetic peptide. </span></p>',
'label1' => 'Validation Data',
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<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIP-a.png" alt="H4K20me3 Antibody ChIP Grade" /></p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIP-b.png" alt="H4K20me3 Antibody for ChIP" /></p>
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<div class="small-6 columns">
<p><small><strong>Figure 1. ChIP results obtained with the Diagenode antibody directed against H4K20me3</strong><br />ChIP assays were performed using human HeLa cells, the Diagenode antibody against H4K20me3 (Cat. No. C15410207) and optimized PCR primer pairs for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (Cat. No. C01010051), using sheared chromatin from 1 million cells. The chromatin was spiked with a panel of in vitro assembled nucleosomes, each containing a specific lysine methylation (SNAP-ChIP K-MetStat Panel, Epicypher). A titration consisting of 0.2, 0.5, 1 and 2 µg of antibody per ChIP experiment was analyzed. IgG (1 µg/IP) was used as a negative IP control.<br /> <strong>Figure 1A.</strong> Quantitative PCR was performed with primers specific for the promoter of the active GAPDH and EIF4A2 genes, used as negative controls, and for the ZNF10 gene and the Sat2 satellite repeat, used as positive controls. The graph shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).<br /> <strong>Figure 1B.</strong> Recovery of the nucleosomes carrying the H4K20me1, H4K20me2, H4K2me3 and the unmodified H4K20 as determined by qPCR. The figure clearly shows the antibody is very specific in ChIP for the H4K20me3 modification. </small></p>
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<p>A. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-A.png" alt="H4K20me3 Antibody for ChIP assay" /></p>
</div>
<div class="small-6 columns">
<p><small><strong> Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H4K20me3</strong><br /> ChIP was performed with 0.5 µg of the Diagenode antibody against H4K20me3 (Cat. No. C15410207) on sheared chromatin from 100,000 K562 cells using the “iDeal ChIP-seq” kit. The IP’d DNA was analysed by QPCR as described above (figure 2A). The IP’d DNA was subsequently analysed on an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. Figure 2B shows the signal distribution along the long arm of chromosome 19 and a zoomin to an enriched region containing several ZNF repeat genes. Figure 2C and D show the enrichment in the telomeric region of chromosome 12, also containing several ZNF repeat genes, and at ZNF510 on chromosome 9, respectively. The position of the amplicon used for ChIP-qPCR is indicated by an arrow. </small></p>
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<div class="row">
<div class="small-12 columns">
<p class="text-center">B. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-B.png" alt="H4K20me3 Antibody ChIP-seq Grade" /></p>
<p class="text-center">C. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-C.png" alt="H4K20me3 Antibody for ChIP-seq" /></p>
<p class="text-center">D. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-D.png" alt="H4K20me3 Antibody validated in ChIP-seq" /></p>
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<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ELISA.png" alt="H4K20me3 Antibody ELISA validation" /></p>
</div>
<div class="small-6 columns">
<p><small><strong> Figure 3. Determination of the antibody titer</strong><br /> To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H4K20me3 (Cat. No. C15410207) in antigen coated wells. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the antibody was estimated to be 1:21,700. </small></p>
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<div class="row">
<div class="small-4 columns">
<p>A. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_CrossReactivity-A.png" alt="H4K20me3 Antibody Dot Blot validation" /></p>
<p>B. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_CrossReactivity-B.png" alt="H4K20me3 Antibody validated in Peptide Array " /></p>
</div>
<div class="small-8 columns">
<p><small><strong>Figure 4. Cross reactivity tests using the Diagenode antibody directed against H4K20me3</strong><br /> Figure 4A To test the cross reactivity of the Diagenode antibody against H4K20me3 (Cat. No. C15410207), a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H4K20. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 4A shows a high specificity of the antibody for the modification of interest. Figure 4B The specificity of the antibody was further demonstrated by peptide array analyses on an array containing 384 peptides with different combinations of modifications from histone H3, H4, H2A and H2B. The antibody was used at a dilution of 1:10,000. Figure 4B shows the specificity factor, calculated as the ratio of the average intensity of all spots containing the mark, divided by the average intensity of all spots not containing the mark. </small></p>
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<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_WB.png" alt="H4K20me3 Antibody validated in Western Blot" /></p>
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<div class="small-8 columns">
<p><small><strong> Figure 5. Western blot analysis using the Diagenode antibody directed against H4K20me3</strong><br /> Western blot was performed on whole cell (25 µg, lane 1) and histone extracts (15 µg, lane 2) from HeLa cells, and on 1 µg of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the Diagenode antibody against H4K20me3 (Cat. No. C15410207). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left. </small></p>
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</div>
<div class="row">
<div class="small-12 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_IF.png" alt="H4K20me3 Antibody validated in Immunofluorescence" /></p>
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<div class="row">
<div class="small-12 columns">
<p><small><strong> Figure 6. Immunofluorescence using the Diagenode antibody directed against H4K20me3</strong><br /> HeLa cells were stained with the Diagenode antibody against H4K20me3 (Cat. No. C15410207) and with DAPI. Cells were fixed with methanol and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H4K20me3 antibody (left) diluted 1:500 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right. </small></p>
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<tr>
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<th>References</th>
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<td>ChIP/ChIP-seq <sup>*</sup></td>
<td>0.5-1 µg per IP</td>
<td>Fig 1, 2</td>
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<tr>
<td>ELISA</td>
<td>1:500</td>
<td>Fig 3</td>
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<tr>
<td>Dot Blotting/Peptide array</td>
<td>1:20,000/1:10,000</td>
<td>Fig 4</td>
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<tr>
<td>Western Blotting</td>
<td>1:1,000</td>
<td>Fig 5</td>
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<tr>
<td>Immunofluorescence</td>
<td>1:500</td>
<td>Fig 6</td>
</tr>
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<p></p>
<p><small><sup>*</sup> Please note that the optimal antibody amount per IP should be determined by the end-user. We recommend testing 0.5-5 µg per IP.</small></p>',
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'name' => 'H4K20me3 polyclonal antibody ',
'description' => '<p><span>Polyclonal antibody raised in rabbit against the region of histone H4 containing the trimethylated lysine 20 (H4K20me3), using a KLH-conjugated synthetic peptide. </span></p>',
'label1' => 'Validation Data',
'info1' => '<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIP-a.png" alt="H4K20me3 Antibody ChIP Grade" /></p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIP-b.png" alt="H4K20me3 Antibody for ChIP" /></p>
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<div class="small-6 columns">
<p><small><strong>Figure 1. ChIP results obtained with the Diagenode antibody directed against H4K20me3</strong><br />ChIP assays were performed using human HeLa cells, the Diagenode antibody against H4K20me3 (Cat. No. C15410207) and optimized PCR primer pairs for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (Cat. No. C01010051), using sheared chromatin from 1 million cells. The chromatin was spiked with a panel of in vitro assembled nucleosomes, each containing a specific lysine methylation (SNAP-ChIP K-MetStat Panel, Epicypher). A titration consisting of 0.2, 0.5, 1 and 2 µg of antibody per ChIP experiment was analyzed. IgG (1 µg/IP) was used as a negative IP control.<br /> <strong>Figure 1A.</strong> Quantitative PCR was performed with primers specific for the promoter of the active GAPDH and EIF4A2 genes, used as negative controls, and for the ZNF10 gene and the Sat2 satellite repeat, used as positive controls. The graph shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).<br /> <strong>Figure 1B.</strong> Recovery of the nucleosomes carrying the H4K20me1, H4K20me2, H4K2me3 and the unmodified H4K20 as determined by qPCR. The figure clearly shows the antibody is very specific in ChIP for the H4K20me3 modification. </small></p>
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<p>A. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-A.png" alt="H4K20me3 Antibody for ChIP assay" /></p>
</div>
<div class="small-6 columns">
<p><small><strong> Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H4K20me3</strong><br /> ChIP was performed with 0.5 µg of the Diagenode antibody against H4K20me3 (Cat. No. C15410207) on sheared chromatin from 100,000 K562 cells using the “iDeal ChIP-seq” kit. The IP’d DNA was analysed by QPCR as described above (figure 2A). The IP’d DNA was subsequently analysed on an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. Figure 2B shows the signal distribution along the long arm of chromosome 19 and a zoomin to an enriched region containing several ZNF repeat genes. Figure 2C and D show the enrichment in the telomeric region of chromosome 12, also containing several ZNF repeat genes, and at ZNF510 on chromosome 9, respectively. The position of the amplicon used for ChIP-qPCR is indicated by an arrow. </small></p>
</div>
</div>
<div class="row">
<div class="small-12 columns">
<p class="text-center">B. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-B.png" alt="H4K20me3 Antibody ChIP-seq Grade" /></p>
<p class="text-center">C. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-C.png" alt="H4K20me3 Antibody for ChIP-seq" /></p>
<p class="text-center">D. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-D.png" alt="H4K20me3 Antibody validated in ChIP-seq" /></p>
</div>
</div>
<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ELISA.png" alt="H4K20me3 Antibody ELISA validation" /></p>
</div>
<div class="small-6 columns">
<p><small><strong> Figure 3. Determination of the antibody titer</strong><br /> To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H4K20me3 (Cat. No. C15410207) in antigen coated wells. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the antibody was estimated to be 1:21,700. </small></p>
</div>
</div>
<div class="row">
<div class="small-4 columns">
<p>A. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_CrossReactivity-A.png" alt="H4K20me3 Antibody Dot Blot validation" /></p>
<p>B. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_CrossReactivity-B.png" alt="H4K20me3 Antibody validated in Peptide Array " /></p>
</div>
<div class="small-8 columns">
<p><small><strong>Figure 4. Cross reactivity tests using the Diagenode antibody directed against H4K20me3</strong><br /> Figure 4A To test the cross reactivity of the Diagenode antibody against H4K20me3 (Cat. No. C15410207), a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H4K20. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 4A shows a high specificity of the antibody for the modification of interest. Figure 4B The specificity of the antibody was further demonstrated by peptide array analyses on an array containing 384 peptides with different combinations of modifications from histone H3, H4, H2A and H2B. The antibody was used at a dilution of 1:10,000. Figure 4B shows the specificity factor, calculated as the ratio of the average intensity of all spots containing the mark, divided by the average intensity of all spots not containing the mark. </small></p>
</div>
</div>
<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_WB.png" alt="H4K20me3 Antibody validated in Western Blot" /></p>
</div>
<div class="small-8 columns">
<p><small><strong> Figure 5. Western blot analysis using the Diagenode antibody directed against H4K20me3</strong><br /> Western blot was performed on whole cell (25 µg, lane 1) and histone extracts (15 µg, lane 2) from HeLa cells, and on 1 µg of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the Diagenode antibody against H4K20me3 (Cat. No. C15410207). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left. </small></p>
</div>
</div>
<div class="row">
<div class="small-12 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_IF.png" alt="H4K20me3 Antibody validated in Immunofluorescence" /></p>
</div>
</div>
<div class="row">
<div class="small-12 columns">
<p><small><strong> Figure 6. Immunofluorescence using the Diagenode antibody directed against H4K20me3</strong><br /> HeLa cells were stained with the Diagenode antibody against H4K20me3 (Cat. No. C15410207) and with DAPI. Cells were fixed with methanol and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H4K20me3 antibody (left) diluted 1:500 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right. </small></p>
</div>
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'slug' => 'h4k20me3-polyclonal-antibody-premium-50-mg-53-ml',
'meta_title' => 'H4K20me3 polyclonal antibody - Premium',
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'host' => '*****',
'id' => '80',
'name' => 'H4K20me3 polyclonal antibody',
'description' => 'Histones are the main constituents of the protein part of chromosomes of eukaryotic cells. They are rich in the amino acids arginine and lysine and have been greatly conserved during evolution. Histones pack the DNA into tight masses of chromatin. Two core histones of each class H2A, H2B, H3 and H4 assemble and are wrapped by 146 base pairs of DNA to form one octameric nucleosome. Histone tails undergo numerous post-translational modifications, which either directly or indirectly alter chromatin structure to facilitate transcriptional activation or repression or other nuclear processes. In addition to the genetic code, combinations of the different histone modifications reveal the so-called “histone code”. Histone methylation and demethylation is dynamically regulated by respectively histone methyl transferases and histone demethylases. Trimethylation of histone H4K20 is associated with inactive genomic regions, satellite repeats and ZNF gene repeats.',
'clonality' => '',
'isotype' => '',
'lot' => 'A2730P',
'concentration' => '0.94 µg/µl',
'reactivity' => 'Human, mouse, wide range expected',
'type' => 'Polyclonal',
'purity' => 'Affinity purified polyclonal antibody.',
'classification' => 'Premium',
'application_table' => '<table>
<thead>
<tr>
<th>Applications</th>
<th>Suggested dilution</th>
<th>References</th>
</tr>
</thead>
<tbody>
<tr>
<td>ChIP/ChIP-seq <sup>*</sup></td>
<td>0.5-1 µg per IP</td>
<td>Fig 1, 2</td>
</tr>
<tr>
<td>ELISA</td>
<td>1:500</td>
<td>Fig 3</td>
</tr>
<tr>
<td>Dot Blotting/Peptide array</td>
<td>1:20,000/1:10,000</td>
<td>Fig 4</td>
</tr>
<tr>
<td>Western Blotting</td>
<td>1:1,000</td>
<td>Fig 5</td>
</tr>
<tr>
<td>Immunofluorescence</td>
<td>1:500</td>
<td>Fig 6</td>
</tr>
</tbody>
</table>
<p></p>
<p><small><sup>*</sup> Please note that the optimal antibody amount per IP should be determined by the end-user. We recommend testing 0.5-5 µg per IP.</small></p>',
'storage_conditions' => 'Store at -20°C; for long storage, store at -80°C. Avoid multiple freeze-thaw cycles.',
'storage_buffer' => 'PBS containing 0.05% azide and 0.05% ProClin 300.',
'precautions' => 'This product is for research use only. Not for use in diagnostic or therapeutic procedures.',
'uniprot_acc' => '',
'slug' => '',
'meta_keywords' => '',
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'modified' => '2020-02-25 13:33:22',
'created' => '0000-00-00 00:00:00',
'select_label' => '80 - H4K20me3 polyclonal antibody (A2730P - 0.94 µg/µl - Human, mouse, wide range expected - Affinity purified polyclonal antibody. - Rabbit)'
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'id' => '41',
'name' => 'C15410207',
'product_id' => '2284',
'modified' => '2016-02-18 20:47:29',
'created' => '2016-02-18 20:47:29'
)
),
'Group' => array(
'Group' => array(
'id' => '41',
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'created' => '2016-02-18 20:47:29'
),
'Master' => array(
'id' => '2284',
'antibody_id' => '80',
'name' => 'H4K20me3 Antibody',
'description' => '<p><span>Polyclonal antibody raised in rabbit against the region of histone <strong>H4 containing the trimethylated lysine 20 (H4K20me3)</strong>, using a KLH-conjugated synthetic peptide. </span></p>',
'label1' => 'Validation data',
'info1' => '<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIP-a.png" alt="H4K20me3 Antibody ChIP Grade" /></p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIP-b.png" alt="H4K20me3 Antibody for ChIP" /></p>
</div>
<div class="small-6 columns">
<p><small><strong>Figure 1. ChIP results obtained with the Diagenode antibody directed against H4K20me3</strong><br />ChIP assays were performed using human HeLa cells, the Diagenode antibody against H4K20me3 (Cat. No. C15410207) and optimized PCR primer pairs for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (Cat. No. C01010051), using sheared chromatin from 1 million cells. The chromatin was spiked with a panel of in vitro assembled nucleosomes, each containing a specific lysine methylation (SNAP-ChIP K-MetStat Panel, Epicypher). A titration consisting of 0.2, 0.5, 1 and 2 µg of antibody per ChIP experiment was analyzed. IgG (1 µg/IP) was used as a negative IP control.<br /> <strong>Figure 1A.</strong> Quantitative PCR was performed with primers specific for the promoter of the active GAPDH and EIF4A2 genes, used as negative controls, and for the ZNF10 gene and the Sat2 satellite repeat, used as positive controls. The graph shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).<br /> <strong>Figure 1B.</strong> Recovery of the nucleosomes carrying the H4K20me1, H4K20me2, H4K2me3 and the unmodified H4K20 as determined by qPCR. The figure clearly shows the antibody is very specific in ChIP for the H4K20me3 modification. </small></p>
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<div class="row">
<div class="small-6 columns">
<p>A. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-A.png" alt="H4K20me3 Antibody for ChIP assay" /></p>
</div>
<div class="small-6 columns">
<p><small><strong> Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H4K20me3</strong><br /> ChIP was performed with 0.5 µg of the Diagenode antibody against H4K20me3 (Cat. No. C15410207) on sheared chromatin from 100,000 K562 cells using the “iDeal ChIP-seq” kit. The IP’d DNA was analysed by QPCR as described above (figure 2A). The IP’d DNA was subsequently analysed on an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. Figure 2B shows the signal distribution along the long arm of chromosome 19 and a zoomin to an enriched region containing several ZNF repeat genes. Figure 2C and D show the enrichment in the telomeric region of chromosome 12, also containing several ZNF repeat genes, and at ZNF510 on chromosome 9, respectively. The position of the amplicon used for ChIP-qPCR is indicated by an arrow. </small></p>
</div>
</div>
<div class="row">
<div class="small-12 columns">
<p class="text-center">B. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-B.png" alt="H4K20me3 Antibody ChIP-seq Grade" /></p>
<p class="text-center">C. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-C.png" alt="H4K20me3 Antibody for ChIP-seq" /></p>
<p class="text-center">D. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-D.png" alt="H4K20me3 Antibody validated in ChIP-seq" /></p>
</div>
</div>
<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ELISA.png" alt="H4K20me3 Antibody ELISA validation" /></p>
</div>
<div class="small-6 columns">
<p><small><strong> Figure 3. Determination of the antibody titer</strong><br /> To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H4K20me3 (Cat. No. C15410207) in antigen coated wells. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the antibody was estimated to be 1:21,700. </small></p>
</div>
</div>
<div class="row">
<div class="small-4 columns">
<p>A. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_CrossReactivity-A.png" alt="H4K20me3 Antibody Dot Blot validation" /></p>
<p>B. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_CrossReactivity-B.png" alt="H4K20me3 Antibody validated in Peptide Array " /></p>
</div>
<div class="small-8 columns">
<p><small><strong>Figure 4. Cross reactivity tests using the Diagenode antibody directed against H4K20me3</strong><br /> Figure 4A To test the cross reactivity of the Diagenode antibody against H4K20me3 (Cat. No. C15410207), a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H4K20. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 4A shows a high specificity of the antibody for the modification of interest. Figure 4B The specificity of the antibody was further demonstrated by peptide array analyses on an array containing 384 peptides with different combinations of modifications from histone H3, H4, H2A and H2B. The antibody was used at a dilution of 1:10,000. Figure 4B shows the specificity factor, calculated as the ratio of the average intensity of all spots containing the mark, divided by the average intensity of all spots not containing the mark. </small></p>
</div>
</div>
<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_WB.png" alt="H4K20me3 Antibody validated in Western Blot" /></p>
</div>
<div class="small-8 columns">
<p><small><strong> Figure 5. Western blot analysis using the Diagenode antibody directed against H4K20me3</strong><br /> Western blot was performed on whole cell (25 µg, lane 1) and histone extracts (15 µg, lane 2) from HeLa cells, and on 1 µg of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the Diagenode antibody against H4K20me3 (Cat. No. C15410207). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left. </small></p>
</div>
</div>
<div class="row">
<div class="small-12 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_IF.png" alt="H4K20me3 Antibody validated in Immunofluorescence" /></p>
</div>
</div>
<div class="row">
<div class="small-12 columns">
<p><small><strong> Figure 6. Immunofluorescence using the Diagenode antibody directed against H4K20me3</strong><br /> HeLa cells were stained with the Diagenode antibody against H4K20me3 (Cat. No. C15410207) and with DAPI. Cells were fixed with methanol and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H4K20me3 antibody (left) diluted 1:500 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right. </small></p>
</div>
</div>',
'label2' => 'Target Description',
'info2' => '<p>Histones are the main constituents of the protein part of chromosomes of eukaryotic cells. They are rich in the amino acids arginine and lysine and have been greatly conserved during evolution. Histones pack the DNA into tight masses of chromatin. Two core histones of each class H2A, H2B, H3 and H4 assemble and are wrapped by 146 base pairs of DNA to form one octameric nucleosome. Histone tails undergo numerous post-translational modifications, which either directly or indirectly alter chromatin structure to facilitate transcriptional activation or repression or other nuclear processes. In addition to the genetic code, combinations of the different histone modifications reveal the so-called “histone code”. Histone methylation and demethylation is dynamically regulated by respectively histone methyl transferases and histone demethylases. Trimethylation of histone H4K20 is associated with inactive genomic regions, satellite repeats and ZNF gene repeats.</p>',
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'type' => 'FRE',
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'price_AUD' => '1175',
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'slug' => 'h4k20me3-polyclonal-antibody-premium-50-mg-53-ml',
'meta_title' => 'H4K20me3 Antibody - ChIP-seq Grade (C15410207) | Diagenode',
'meta_keywords' => '',
'meta_description' => 'H4K20me3 (Histone H4 trimethylated at lysine 20) Polyclonal Antibody validated in ChIP-seq, ChIP-qPCR, ELISA, DB, WB and IF. Specificity confirmed by Peptide array assay. Batch-specific data available on the website. Sample size available.',
'modified' => '2024-01-17 16:52:00',
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'Related' => array(
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'id' => '2914',
'antibody_id' => null,
'name' => 'Auto ChIPmentation Kit for Histones - Replaced by C01011009',
'description' => '<div class="extra-spaced"><center></center></div>
<div class="row">
<div class="small-12 medium-8 large-8 columns"><!--<div class="extra-spaced"><center><a href="https://www.diagenode.com/en/pages/grant"><img src="https://www.diagenode.com/img/banners/banner-chipmentation-grant-580x120.jpg" /></a></center></div>-->
<div align="center"><video width="400" height="250" autoplay="autoplay" muted="" loop="loop" controls="controls" src="https://www.diagenode.com/videos/chipmentation-dgo.mp4" frameborder="0" allowfullscreen="allowfullscreen"></video></div>
<div align="center"><a href="https://www.diagenode.com/pages/form-chipmentation" class="center alert radius button"><i class="fa fa-info"></i> Contact us</a></div>
</div>
<div class="small-12 medium-4 large-4 columns"><center><a href="https://www.citeab.com/awards/2018/innovative-product-of-the-year" target="_blank"><img src="https://www.diagenode.com/emailing/images/citeab.png" width="224" height="200" /></a></center></div>
</div>
<p></p>
<p><strong>This product must be used with the <a href="https://www.diagenode.com/en/p/sx-8g-ip-star-compact-automated-system-1-unit">IP-Star Compact Automated System</a>.</strong></p>
<p>Diagenode’s latest technology for histone ChIP-seq, ChIPmentation, incorporates automation with an ligation-free protocol that integrates chromatin immunoprecipitation with NGS library preparation. Using histone ChIPmentation, you can avoid the multi-step ligation required for traditional histone ChIP-seq protocols, providing an easier protocol, faster time to results, and unmatched efficiency and reproducibility for histone ChIP-seq from challenging and low input samples. The ChIPmentaion Kit for Histones is a complete solution, including all buffers for chromatin preparation, immunoprecipitation, tagmentation and multiplexing using 24 single indexes.</p>',
'label1' => 'Characteristics',
'info1' => '<p>ChIPmentation is based on tagmentation that allows library preparation to be integrated during the ChIP itself using transposase and sequencing-compatible adaptors. ChIPmentation involves much easier and shorter protocol with high efficiency for low input samples using optimized ChIP and NGS sample preparation reagents on the IP-Star Compact Automated System. The IP-Star Compact Automated System ensures the highest reproducibility for challenging samples from limited input amounts.</p>
<h3>Benefits of the ChIPmentation system for histone ChIP-seq</h3>
<ul>
<li>Automates ChIP-seq of histones on the IP-Star® for highest reproducibility</li>
<li>Easier and faster than classical ChIP-seq</li>
<li>Validated for various histone marks</li>
<li>Ideal for analysis of large cohorts of samples</li>
<li>Ideal for analysis of large number of marks on a unique sample</li>
<li>High quality sequencing data</li>
</ul>
<p><img src="https://www.diagenode.com/img/product/chipmentation/chipmentation.jpg" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<h3>ChIPmentation - Highest reliability for even the lowest inputs</h3>
<p>The new Auto ChIPmentation technology has been tested on histone marks and compared to available datasets from the ENCODE project (Figure 1). ChIPmentation generated high quality data with low background. In addition, more than 99% of the top 40% peaks obtained with auto-ChIPmentation overlap with ENCODE datasets, which shows that ChIP-seq data obtained with ChIPmentation are highly reliable.</p>
<div class="spaced"><center>A.<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/auto-chipmentation.png" alt="Diagenode-Auto-ChIPmentation" /></center><br /><center>B. Match of the Top40 peaks of Auto-ChIPmentation<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/match-of-the-top40-peaks-of-auto-chipmentation.png" /></center></div>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure1"><i class="fa fa-caret-right"></i> Figure 1</a>
<div class="content" id="figure1">
<p><strong>Comparison of ChIPmentation sequencing results and reference ChIP-seq data.</strong><br /> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 100.000 cells was used for the immunoprecipitation with the Diagenode antibody targeting H3K4me3 (Cat. no. C15410003). The experiment was performed on the IP-Star® Compact Automated System. A. Distribution of the ChIPmentation and ENCODE readsets in a representative region of the genome. B. Comparison of the top 40% peaks from ChIPmentation dataset to the ENCODE dataset.</p>
</div>
</li>
</ul>
<p class="extra-spaced">As ChIPmentation is a ligation-free protocol and requires fewer steps than a classical workflow, it allows the generation of excellent ChIP-seq data from reduced starting amounts. The sequencing data generated from 5,000 and 10,000 cells are very similar to those obtained with 100,000 cells, showing more than 98% overlap of the top 40% peaks (Figure 2).</p>
<center>A.<br /><img class="extra-spaced" style="margin-bottom: 20px;" src="https://www.diagenode.com/img/categories/kits_chromatin_function/chipmentation-sequencing-figure-a.jpg" alt="Diagenode-ChIPmentation-sequencing" /></center>
<div class="row">
<div class="small-6 columns" style="text-align: left;">B. Match of the Top40 peaks of ChIPmentation from 10.000 cells<br /><center><img src="https://www.diagenode.com/img/categories/kits_chromatin_function/match-of-the-top40-peaks-of-chipmentation-from-cent-cells.png" /></center></div>
<div class="small-6 columns">C. Match of the Top40 peaks of ChIPmentation from 5.000 cells<center><img src="https://www.diagenode.com/img/categories/kits_chromatin_function/match-of-the-top40-peaks-of-chipmentation-from-five-cells.png" /></center></div>
</div>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure2"><i class="fa fa-caret-right"></i> Figure 2</a>
<div class="content" id="figure2">
<p><strong>ChIPmentation sequencing results obtained from decreasing starting amounts of cells.</strong><br /> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 100.000, 10.000 and 5.000 cells was used for the immunoprecipitation with the Diagenode antibody targeting H3K4me3 (Cat. no. C15410003). The experiments were performed on the IP-Star® Compact Automated System. A. Distribution of the ChIPmentation readsets in a representative region of the genome. B. and C. Comparison of the top 40% peaks from 10.000 (B.) and 5.000 (C.) cells with dataset generated from 100.000 cells.</p>
</div>
</li>
</ul>
<p class="extra-spaced">Moreover, in order to check the reproducibility of data generated with low cell amounts, correlation between replicates for 10.000 and 5.000 starting cells was performed (Figure 3). The data show very high correlations with Pearson’s coefficients of 0.96 for both conditions.</p>
<div class="row">
<div class="small-6 columns" style="text-align: left;"><center>A. Auto ChIPmentation on 10.000 cells<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/auto-chipmentation-on-10000cells.jpg" caption="false" width="480" height="420" /></center></div>
<div class="small-6 columns"><center>B. Auto ChIPmentation on 5.000 cells<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/auto-chipmentation-on-5000cells.jpg" width="480" height="420" /></center></div>
</div>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure3"><i class="fa fa-caret-right"></i> Figure 3</a>
<div class="content" id="figure3">
<p><strong>Correlation of replicates of auto-ChIPmentation experiments.</strong> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 10.000 and 5.000 cells was used for the immunoprecipitation with the Diagenode antibody targeting H3K4me3 (Cat. no. C15410003). The experiments were performed on the IP-Star® Compact Automated System.The scatterplots show the correlation between the enrichments between two replicates. The green line represents an ideal case where the two replicates are identical, while the red curve fitted on the data points by a LOESS regression shows the actual trend. A. Correlation analysis between two replicates of auto-ChIPmentation experiments starting from 10.000 cells. B. Correlation analysis between two replicates of auto-ChIPmentation experiments starting from 5.000 cells.</p>
</div>
</li>
</ul>
<p>The Auto-ChIPmentation workflow is easy and fast, producing high-quality ChIP-seq data even on low starting amounts.</p>
<center><img src="https://www.diagenode.com/img/product/chipmentation/chipmentation-sequencing-results-for-histone.jpg" width="678" height="240" /></center>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure4"><i class="fa fa-caret-right"></i> Figure 4</a>
<div class="content" id="figure4">
<p><strong>ChIPmentation sequencing results for four histone marks.</strong> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 1,000,000 cells was used for the immunoprecipitation with the Diagenode antibodies targeting H3K4me1 (Cat. no. C15410194), H3K27me3 (Cat. no. C15410195), H3K27ac (Cat. no. C15410196) and IgG (Cat. no. C15410206). The experiment was performed on the IP-Star® Compact Automated System.</p>
</div>
</li>
</ul>',
'label2' => 'Additional solutions compatible with Auto ChIPmentation Kit for Histones',
'info2' => '<p><a href="https://www.diagenode.com/en/p/chromatin-shearing-optimization-kit-low-sds-100-million-cells">Chromatin shearing optimization kit - Low SDS (iDeal Kit for Histones)</a> optimizes chromatin shearing, a critical step for ChIP.</p>
<p><a href="https://www.diagenode.com/en/categories/chip-seq-grade-antibodies">ChIP-seq grade anti-histone antibodies</a> provide high yields with excellent specificity and sensitivity</p>',
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<div class="small-12 medium-12 large-12 columns">Enzyme-linked immunosorbent assay.</div>
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'description' => '<p><strong>Western blot</strong> : The quality of antibodies used in this technique is crucial for correct and specific protein identification. Diagenode offers huge selection of highly sensitive and specific western blot-validated antibodies.</p>
<p>Learn more about: <a href="https://www.diagenode.com/applications/western-blot">Loading control, MW marker visualization</a><em>. <br /></em></p>
<p><em></em>Check our selection of antibodies validated in Western blot.</p>',
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'description' => '<p><strong>Immunofluorescence</strong>:</p>
<p>Diagenode offers huge selection of highly sensitive antibodies validated in IF.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15200229-IF.jpg" alt="" height="245" width="256" /></p>
<p><sup><strong>Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong></sup></p>
<p><sup>HeLa cells transfected with a Cas9 expression vector (left) or untransfected cells (right) were fixed in methanol at -20°C, permeabilized with acetone at -20°C and blocked with PBS containing 2% BSA. The cells were stained with the Cas9 C-terminal antibody (Cat. No. C15200229) diluted 1:400, followed by incubation with an anti-mouse secondary antibody coupled to AF488. The bottom images show counter-staining of the nuclei with Hoechst 33342.</sup></p>
<h5><sup>Check our selection of antibodies validated in IF.</sup></h5>',
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'meta_description' => 'Diagenode offers a wide range of antibodies and technical support for ChIP Sequencing applications',
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'description' => '<p>Histones are the main protein components of chromatin involved in the compaction of DNA into nucleosomes, the basic units of chromatin. A <strong>nucleosome</strong> consists of one pair of each of the core histones (<strong>H2A</strong>, <strong>H2B</strong>, <strong>H3</strong> and <strong>H4</strong>) forming an octameric structure wrapped by 146 base pairs of DNA. The different nucleosomes are linked by the linker histone<strong> H1, </strong>allowing for further condensation of chromatin.</p>
<p>The core histones have a globular structure with large unstructured N-terminal tails protruding from the nucleosome. They can undergo to multiple post-translational modifications (PTM), mainly at the N-terminal tails. These <strong>post-translational modifications </strong>include methylation, acetylation, phosphorylation, ubiquitinylation, citrullination, sumoylation, deamination and crotonylation. The most well characterized PTMs are <strong>methylation,</strong> <strong>acetylation and phosphorylation</strong>. Histone methylation occurs mainly on lysine (K) residues, which can be mono-, di- or tri-methylated, and on arginines (R), which can be mono-methylated and symmetrically or asymmetrically di-methylated. Histone acetylation occurs on lysines and histone phosphorylation mainly on serines (S), threonines (T) and tyrosines (Y).</p>
<p>The PTMs of the different residues are involved in numerous processes such as DNA repair, DNA replication and chromosome condensation. They influence the chromatin organization and can be positively or negatively associated with gene expression. Trimethylation of H3K4, H3K36 and H3K79, and lysine acetylation generally result in an open chromatin configuration (figure below) and are therefore associated with <strong>euchromatin</strong> and gene activation. Trimethylation of H3K9, K3K27 and H4K20, on the other hand, is enriched in <strong>heterochromatin </strong>and associated with gene silencing. The combination of different histone modifications is called the "<strong>histone code</strong>”, analogous to the genetic code.</p>
<p><img src="https://www.diagenode.com/img/categories/antibodies/histone-marks-illustration.png" /></p>
<p>Diagenode is proud to offer a large range of antibodies against histones and histone modifications. Our antibodies are highly specific and have been validated in many applications, including <strong>ChIP</strong> and <strong>ChIP-seq</strong>.</p>
<p>Diagenode’s collection includes antibodies recognizing:</p>
<ul>
<li><strong>Histone H1 variants</strong></li>
<li><strong>Histone H2A, H2A variants and histone H2A</strong> <strong>modifications</strong> (serine phosphorylation, lysine acetylation, lysine ubiquitinylation)</li>
<li><strong>Histone H2B and H2B</strong> <strong>modifications </strong>(serine phosphorylation, lysine acetylation)</li>
<li><strong>Histone H3 and H3 modifications </strong>(lysine methylation (mono-, di- and tri-methylated), lysine acetylation, serine phosphorylation, threonine phosphorylation, arginine methylation (mono-methylated, symmetrically and asymmetrically di-methylated))</li>
<li><strong>Histone H4 and H4 modifications (</strong>lysine methylation (mono-, di- and tri-methylated), lysine acetylation, arginine methylation (mono-methylated and symmetrically di-methylated), serine phosphorylation )</li>
</ul>
<p><span style="font-weight: 400;"><strong>HDAC's HAT's, HMT's and other</strong> <strong>enzymes</strong> which modify histones can be found in the category <a href="../categories/chromatin-modifying-proteins-histone-transferase">Histone modifying enzymes</a><br /></span></p>
<p><span style="font-weight: 400;"> Diagenode’s highly validated antibodies:</span></p>
<ul>
<li><span style="font-weight: 400;"> Highly sensitive and specific</span></li>
<li><span style="font-weight: 400;"> Cost-effective (requires less antibody per reaction)</span></li>
<li><span style="font-weight: 400;"> Batch-specific data is available on the website</span></li>
<li><span style="font-weight: 400;"> Expert technical support</span></li>
<li><span style="font-weight: 400;"> Sample sizes available</span></li>
<li><span style="font-weight: 400;"> 100% satisfaction guarantee</span></li>
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'meta_description' => 'Polyclonal and Monoclonal Antibodies against Histones and their modifications validated for many applications, including Chromatin Immunoprecipitation (ChIP) and ChIP-Sequencing (ChIP-seq)',
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'description' => '<p><b>Unparalleled ChIP-Seq results with the most rigorously validated antibodies</b></p>
<p><span style="font-weight: 400;">Diagenode provides leading solutions for epigenetic research. Because ChIP-seq is a widely-used technique, we validate our antibodies in ChIP and ChIP-seq experiments (in addition to conventional methods like Western blot, Dot blot, ELISA, and immunofluorescence) to provide the highest quality antibody. We standardize our validation and production to guarantee high product quality without technical bias. Diagenode guarantees ChIP-seq grade antibody performance under our suggested conditions.</span></p>
<div class="row">
<div class="small-12 medium-9 large-9 columns">
<p><strong>ChIP-seq profile</strong> of active (H3K4me3 and H3K36me3) and inactive (H3K27me3) marks using Diagenode antibodies.</p>
<img src="https://www.diagenode.com/img/categories/antibodies/chip-seq-grade-antibodies.png" /></div>
<div class="small-12 medium-3 large-3 columns">
<p><small> ChIP was performed on sheared chromatin from 100,000 K562 cells using iDeal ChIP-seq kit for Histones (cat. No. C01010051) with 1 µg of the Diagenode antibodies against H3K27me3 (cat. No. C15410195) and H3K4me3 (cat. No. C15410003), and 0.5 µg of the antibody against H3K36me3 (cat. No. C15410192). The IP'd DNA was subsequently analysed on an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer's instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. The figure shows the signal distribution along the complete sequence of human chromosome 3, a zoomin to a 10 Mb region and a further zoomin to a 1.5 Mb region. </small></p>
</div>
</div>
<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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'meta_title' => 'Diagenode's selection of Antibodies is exclusively dedicated for Epigenetic Research | Diagenode',
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'description' => '<div class="row">
<div class="small-12 columns"><center></center>
<p><br />Chromatin immunoprecipitation (<b>ChIP</b>) is a technique to study the associations of proteins with the specific genomic regions in intact cells. One of the most important steps of this protocol is the immunoprecipitation of targeted protein using the antibody specifically recognizing it. The quality of antibodies used in ChIP is essential for the success of the experiment. Diagenode offers extensively validated ChIP-grade antibodies, confirmed for their specificity, and high level of performance in ChIP. Each batch is validated, and batch-specific data are available on the website.</p>
<p></p>
</div>
</div>
<p><strong>ChIP results</strong> obtained with the antibody directed against H3K4me3 (Cat. No. <a href="../p/h3k4me3-polyclonal-antibody-premium-50-ug-50-ul">C15410003</a>). </p>
<div class="row">
<div class="small-12 medium-6 large-6 columns"><img src="https://www.diagenode.com/img/product/antibodies/C15410003-fig1-ChIP.jpg" alt="" width="400" height="315" /> </div>
<div class="small-12 medium-6 large-6 columns">
<p></p>
<p></p>
<p></p>
</div>
</div>
<p></p>
<p>Our aim at Diagenode is to offer the largest collection of highly specific <strong>ChIP-grade antibodies</strong>. We add new antibodies monthly. Find your ChIP-grade antibody in the list below and check more information about tested applications, extensive validation data, and product information.</p>',
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'meta_description' => 'Diagenode Offers Extensively Validated ChIP-Grade Antibodies, Confirmed for their Specificity, and high level of Performance in Chromatin Immunoprecipitation ChIP',
'meta_title' => 'Chromatin immunoprecipitation ChIP-grade antibodies | Diagenode',
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'description' => '<p style="text-align: justify;"><span>Epigenetic research tools have evolved over time from endpoint PCR to qPCR to the analyses of large sets of genome-wide sequencing data. ChIP sequencing (ChIP-seq) has now become the gold standard method for chromatin studies, given the accuracy and coverage scale of the approach over other methods. Successful ChIP-seq, however, requires a higher level of experimental accuracy and consistency in all steps of ChIP than ever before. Particularly crucial is the quality of ChIP antibodies. </span></p>',
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'name' => 'The 20S proteasome activator PA28γ controls the compaction of chromatin',
'authors' => 'Didier Fesquet, David Llères, Cristina Viganò, Francisca Méchali, Séverine Boulon, Robert Feil, Olivier Coux, Catherine Bonne-Andrea, Véronique Baldin',
'description' => '<p>The nuclear PA28γ is known to activate the 20S proteasome, but its precise cellular functions remains unclear. Here, we identify PA28γ as a key factor that structures heterochromatin. We find that in human cells, a fraction of PA28γ-20S proteasome complexes localizes within HP1-linked heterochromatin foci. Our biochemical studies show that PA28γ interacts with HP1 proteins, particularly HP1β, which recruits the PA28γ-20S proteasome complexes to heterochromatin. Loss of PA28γ does not modify the localization of HP1β, its mobility within nuclear foci, or the level of H3K9 tri-methylation, but reduces H4K20 mono- and tri-methylation, modifications involved in heterochromatin establishment. Concordantly, using a quantitative FRET-based microscopy assay to monitor nanometer-scale proximity between nucleosomes in living cells, we find that PA28γ regulates nucleosome proximity within heterochromatin, and thereby controls its compaction. This function of PA28γ is independent of the 20S proteasome. Importantly, HP1β on its own is unable to drive heterochromatin compaction without PA28γ. Combined, our data reveal an unexpected chromatin structural role of PA28γ, and provide new insights into the mechanism that controls HP1β-mediated heterochromatin compaction.</p>',
'date' => '2020-05-28',
'pmid' => 'https://www.biorxiv.org/content/10.1101/716332v1.article-info',
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'name' => 'In vitro capture and characterization of embryonic rosette-stage pluripotency between naive and primed states.',
'authors' => 'Neagu A, van Genderen E, Escudero I, Verwegen L, Kurek D, Lehmann J, Stel J, Dirks RAM, van Mierlo G, Maas A, Eleveld C, Ge Y, den Dekker AT, Brouwer RWW, van IJcken WFJ, Modic M, Drukker M, Jansen JH, Rivron NC, Baart EB, Marks H, Ten Berge D',
'description' => '<p>Following implantation, the naive pluripotent epiblast of the mouse blastocyst generates a rosette, undergoes lumenogenesis and forms the primed pluripotent egg cylinder, which is able to generate the embryonic tissues. How pluripotency progression and morphogenesis are linked and whether intermediate pluripotent states exist remain controversial. We identify here a rosette pluripotent state defined by the co-expression of naive factors with the transcription factor OTX2. Downregulation of blastocyst WNT signals drives the transition into rosette pluripotency by inducing OTX2. The rosette then activates MEK signals that induce lumenogenesis and drive progression to primed pluripotency. Consequently, combined WNT and MEK inhibition supports rosette-like stem cells, a self-renewing naive-primed intermediate. Rosette-like stem cells erase constitutive heterochromatin marks and display a primed chromatin landscape, with bivalently marked primed pluripotency genes. Nonetheless, WNT induces reversion to naive pluripotency. The rosette is therefore a reversible pluripotent intermediate whereby control over both pluripotency progression and morphogenesis pivots from WNT to MEK signals.</p>',
'date' => '2020-05-01',
'pmid' => 'http://www.pubmed.gov/32367046',
'doi' => '10.1038/s41556-020-0508-x',
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'name' => 'Myc Regulates Chromatin Decompaction and Nuclear Architecture during B Cell Activation',
'authors' => 'Kieffer-Kwon K.R. et al.',
'description' => '<p>50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input. Single-molecule imaging shows that this step lowers transcription factor residence time and non-specific collisions during sampling for DNA targets. Third, chromatin interactions shift from long range to predominantly short range, and CTCF-mediated loops and contact domains double in numbers. This architectural change facilitates cognate promoter-enhancer contacts and also requires Myc and continual ATP production. Our results thus define the nature and transcriptional impact of chromatin decondensation and reveal an unexpected role for Myc in the establishment of nuclear topology in mammalian cells.</p>',
'date' => '2017-08-17',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/28803781',
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'name' => 'SDS C15410207 H4K20me3 Antibody GB en',
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<div class="row">
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<a href="/jp/p/chipmentation-for-histones-24-rxns"><img src="/img/grey-logo.jpg" alt="default alt" class="th"/></a> </div>
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<div class="small-6 columns" style="padding-left:0px;padding-right:0px;margin-top:-6px;margin-left:-1px">
<span class="success label" style="">C01011000</span>
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<!--a href="#" style="color:#B21329"><i class="fa fa-info-circle"></i></a-->
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<div class="small-12 columns" >
<h6 style="height:60px">Auto ChIPmentation Kit for Histones</h6>
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<div class="small-12 medium-8 large-8 columns"><!--<div class="extra-spaced"><center><a href="https://www.diagenode.com/en/pages/grant"><img src="https://www.diagenode.com/img/banners/banner-chipmentation-grant-580x120.jpg" /></a></center></div>-->
<div align="center"><video width="400" height="250" autoplay="autoplay" muted="" loop="loop" controls="controls" src="https://www.diagenode.com/videos/chipmentation-dgo.mp4" frameborder="0" allowfullscreen="allowfullscreen"></video></div>
<div align="center"><a href="https://www.diagenode.com/pages/form-chipmentation" class="center alert radius button"><i class="fa fa-info"></i> Contact us</a></div>
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<div class="small-12 medium-4 large-4 columns"><center><a href="https://www.citeab.com/awards/2018/innovative-product-of-the-year" target="_blank"><img src="https://www.diagenode.com/emailing/images/citeab.png" width="224" height="200" /></a></center></div>
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<p></p>
<p><strong>This product must be used with the <a href="https://www.diagenode.com/en/p/sx-8g-ip-star-compact-automated-system-1-unit">IP-Star Compact Automated System</a>.</strong></p>
<p>Diagenode’s latest technology for histone ChIP-seq, ChIPmentation, incorporates automation with an ligation-free protocol that integrates chromatin immunoprecipitation with NGS library preparation. Using histone ChIPmentation, you can avoid the multi-step ligation required for traditional histone ChIP-seq protocols, providing an easier protocol, faster time to results, and unmatched efficiency and reproducibility for histone ChIP-seq from challenging and low input samples. The ChIPmentaion Kit for Histones is a complete solution, including all buffers for chromatin preparation, immunoprecipitation, tagmentation and multiplexing using 24 single indexes.</p>',
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'info1' => '<p>ChIPmentation is based on tagmentation that allows library preparation to be integrated during the ChIP itself using transposase and sequencing-compatible adaptors. ChIPmentation involves much easier and shorter protocol with high efficiency for low input samples using optimized ChIP and NGS sample preparation reagents on the IP-Star Compact Automated System. The IP-Star Compact Automated System ensures the highest reproducibility for challenging samples from limited input amounts.</p>
<h3>Benefits of the ChIPmentation system for histone ChIP-seq</h3>
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<p><img src="https://www.diagenode.com/img/product/chipmentation/chipmentation.jpg" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<h3>ChIPmentation - Highest reliability for even the lowest inputs</h3>
<p>The new Auto ChIPmentation technology has been tested on histone marks and compared to available datasets from the ENCODE project (Figure 1). ChIPmentation generated high quality data with low background. In addition, more than 99% of the top 40% peaks obtained with auto-ChIPmentation overlap with ENCODE datasets, which shows that ChIP-seq data obtained with ChIPmentation are highly reliable.</p>
<div class="spaced"><center>A.<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/auto-chipmentation.png" alt="Diagenode-Auto-ChIPmentation" /></center><br /><center>B. Match of the Top40 peaks of Auto-ChIPmentation<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/match-of-the-top40-peaks-of-auto-chipmentation.png" /></center></div>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure1"><i class="fa fa-caret-right"></i> Figure 1</a>
<div class="content" id="figure1">
<p><strong>Comparison of ChIPmentation sequencing results and reference ChIP-seq data.</strong><br /> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 100.000 cells was used for the immunoprecipitation with the Diagenode antibody targeting H3K4me3 (Cat. no. C15410003). The experiment was performed on the IP-Star® Compact Automated System. A. Distribution of the ChIPmentation and ENCODE readsets in a representative region of the genome. B. Comparison of the top 40% peaks from ChIPmentation dataset to the ENCODE dataset.</p>
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<p class="extra-spaced">As ChIPmentation is a ligation-free protocol and requires fewer steps than a classical workflow, it allows the generation of excellent ChIP-seq data from reduced starting amounts. The sequencing data generated from 5,000 and 10,000 cells are very similar to those obtained with 100,000 cells, showing more than 98% overlap of the top 40% peaks (Figure 2).</p>
<center>A.<br /><img class="extra-spaced" style="margin-bottom: 20px;" src="https://www.diagenode.com/img/categories/kits_chromatin_function/chipmentation-sequencing-figure-a.jpg" alt="Diagenode-ChIPmentation-sequencing" /></center>
<div class="row">
<div class="small-6 columns" style="text-align: left;">B. Match of the Top40 peaks of ChIPmentation from 10.000 cells<br /><center><img src="https://www.diagenode.com/img/categories/kits_chromatin_function/match-of-the-top40-peaks-of-chipmentation-from-cent-cells.png" /></center></div>
<div class="small-6 columns">C. Match of the Top40 peaks of ChIPmentation from 5.000 cells<center><img src="https://www.diagenode.com/img/categories/kits_chromatin_function/match-of-the-top40-peaks-of-chipmentation-from-five-cells.png" /></center></div>
</div>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure2"><i class="fa fa-caret-right"></i> Figure 2</a>
<div class="content" id="figure2">
<p><strong>ChIPmentation sequencing results obtained from decreasing starting amounts of cells.</strong><br /> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 100.000, 10.000 and 5.000 cells was used for the immunoprecipitation with the Diagenode antibody targeting H3K4me3 (Cat. no. C15410003). The experiments were performed on the IP-Star® Compact Automated System. A. Distribution of the ChIPmentation readsets in a representative region of the genome. B. and C. Comparison of the top 40% peaks from 10.000 (B.) and 5.000 (C.) cells with dataset generated from 100.000 cells.</p>
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<p class="extra-spaced">Moreover, in order to check the reproducibility of data generated with low cell amounts, correlation between replicates for 10.000 and 5.000 starting cells was performed (Figure 3). The data show very high correlations with Pearson’s coefficients of 0.96 for both conditions.</p>
<div class="row">
<div class="small-6 columns" style="text-align: left;"><center>A. Auto ChIPmentation on 10.000 cells<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/auto-chipmentation-on-10000cells.jpg" caption="false" width="480" height="420" /></center></div>
<div class="small-6 columns"><center>B. Auto ChIPmentation on 5.000 cells<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/auto-chipmentation-on-5000cells.jpg" width="480" height="420" /></center></div>
</div>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure3"><i class="fa fa-caret-right"></i> Figure 3</a>
<div class="content" id="figure3">
<p><strong>Correlation of replicates of auto-ChIPmentation experiments.</strong> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 10.000 and 5.000 cells was used for the immunoprecipitation with the Diagenode antibody targeting H3K4me3 (Cat. no. C15410003). The experiments were performed on the IP-Star® Compact Automated System.The scatterplots show the correlation between the enrichments between two replicates. The green line represents an ideal case where the two replicates are identical, while the red curve fitted on the data points by a LOESS regression shows the actual trend. A. Correlation analysis between two replicates of auto-ChIPmentation experiments starting from 10.000 cells. B. Correlation analysis between two replicates of auto-ChIPmentation experiments starting from 5.000 cells.</p>
</div>
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<p>The Auto-ChIPmentation workflow is easy and fast, producing high-quality ChIP-seq data even on low starting amounts.</p>
<center><img src="https://www.diagenode.com/img/product/chipmentation/chipmentation-sequencing-results-for-histone.jpg" width="678" height="240" /></center>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure4"><i class="fa fa-caret-right"></i> Figure 4</a>
<div class="content" id="figure4">
<p><strong>ChIPmentation sequencing results for four histone marks.</strong> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 1,000,000 cells was used for the immunoprecipitation with the Diagenode antibodies targeting H3K4me1 (Cat. no. C15410194), H3K27me3 (Cat. no. C15410195), H3K27ac (Cat. no. C15410196) and IgG (Cat. no. C15410206). The experiment was performed on the IP-Star® Compact Automated System.</p>
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<p><a href="https://www.diagenode.com/en/categories/chip-seq-grade-antibodies">ChIP-seq grade anti-histone antibodies</a> provide high yields with excellent specificity and sensitivity</p>',
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'name' => 'Myc Regulates Chromatin Decompaction and Nuclear Architecture during B Cell Activation',
'authors' => 'Kieffer-Kwon K.R. et al.',
'description' => '<p>50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input. Single-molecule imaging shows that this step lowers transcription factor residence time and non-specific collisions during sampling for DNA targets. Third, chromatin interactions shift from long range to predominantly short range, and CTCF-mediated loops and contact domains double in numbers. This architectural change facilitates cognate promoter-enhancer contacts and also requires Myc and continual ATP production. Our results thus define the nature and transcriptional impact of chromatin decondensation and reveal an unexpected role for Myc in the establishment of nuclear topology in mammalian cells.</p>',
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'description' => '<p><span>Polyclonal antibody raised in rabbit against the region of histone H4 containing the trimethylated lysine 20 (H4K20me3), using a KLH-conjugated synthetic peptide. </span></p>',
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<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIP-a.png" alt="H4K20me3 Antibody ChIP Grade" /></p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIP-b.png" alt="H4K20me3 Antibody for ChIP" /></p>
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<div class="small-6 columns">
<p><small><strong>Figure 1. ChIP results obtained with the Diagenode antibody directed against H4K20me3</strong><br />ChIP assays were performed using human HeLa cells, the Diagenode antibody against H4K20me3 (Cat. No. C15410207) and optimized PCR primer pairs for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (Cat. No. C01010051), using sheared chromatin from 1 million cells. The chromatin was spiked with a panel of in vitro assembled nucleosomes, each containing a specific lysine methylation (SNAP-ChIP K-MetStat Panel, Epicypher). A titration consisting of 0.2, 0.5, 1 and 2 µg of antibody per ChIP experiment was analyzed. IgG (1 µg/IP) was used as a negative IP control.<br /> <strong>Figure 1A.</strong> Quantitative PCR was performed with primers specific for the promoter of the active GAPDH and EIF4A2 genes, used as negative controls, and for the ZNF10 gene and the Sat2 satellite repeat, used as positive controls. The graph shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).<br /> <strong>Figure 1B.</strong> Recovery of the nucleosomes carrying the H4K20me1, H4K20me2, H4K2me3 and the unmodified H4K20 as determined by qPCR. The figure clearly shows the antibody is very specific in ChIP for the H4K20me3 modification. </small></p>
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<p>A. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-A.png" alt="H4K20me3 Antibody for ChIP assay" /></p>
</div>
<div class="small-6 columns">
<p><small><strong> Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H4K20me3</strong><br /> ChIP was performed with 0.5 µg of the Diagenode antibody against H4K20me3 (Cat. No. C15410207) on sheared chromatin from 100,000 K562 cells using the “iDeal ChIP-seq” kit. The IP’d DNA was analysed by QPCR as described above (figure 2A). The IP’d DNA was subsequently analysed on an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. Figure 2B shows the signal distribution along the long arm of chromosome 19 and a zoomin to an enriched region containing several ZNF repeat genes. Figure 2C and D show the enrichment in the telomeric region of chromosome 12, also containing several ZNF repeat genes, and at ZNF510 on chromosome 9, respectively. The position of the amplicon used for ChIP-qPCR is indicated by an arrow. </small></p>
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<div class="row">
<div class="small-12 columns">
<p class="text-center">B. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-B.png" alt="H4K20me3 Antibody ChIP-seq Grade" /></p>
<p class="text-center">C. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-C.png" alt="H4K20me3 Antibody for ChIP-seq" /></p>
<p class="text-center">D. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-D.png" alt="H4K20me3 Antibody validated in ChIP-seq" /></p>
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<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ELISA.png" alt="H4K20me3 Antibody ELISA validation" /></p>
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<div class="small-6 columns">
<p><small><strong> Figure 3. Determination of the antibody titer</strong><br /> To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H4K20me3 (Cat. No. C15410207) in antigen coated wells. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the antibody was estimated to be 1:21,700. </small></p>
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<div class="row">
<div class="small-4 columns">
<p>A. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_CrossReactivity-A.png" alt="H4K20me3 Antibody Dot Blot validation" /></p>
<p>B. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_CrossReactivity-B.png" alt="H4K20me3 Antibody validated in Peptide Array " /></p>
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<div class="small-8 columns">
<p><small><strong>Figure 4. Cross reactivity tests using the Diagenode antibody directed against H4K20me3</strong><br /> Figure 4A To test the cross reactivity of the Diagenode antibody against H4K20me3 (Cat. No. C15410207), a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H4K20. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 4A shows a high specificity of the antibody for the modification of interest. Figure 4B The specificity of the antibody was further demonstrated by peptide array analyses on an array containing 384 peptides with different combinations of modifications from histone H3, H4, H2A and H2B. The antibody was used at a dilution of 1:10,000. Figure 4B shows the specificity factor, calculated as the ratio of the average intensity of all spots containing the mark, divided by the average intensity of all spots not containing the mark. </small></p>
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<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_WB.png" alt="H4K20me3 Antibody validated in Western Blot" /></p>
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<div class="small-8 columns">
<p><small><strong> Figure 5. Western blot analysis using the Diagenode antibody directed against H4K20me3</strong><br /> Western blot was performed on whole cell (25 µg, lane 1) and histone extracts (15 µg, lane 2) from HeLa cells, and on 1 µg of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the Diagenode antibody against H4K20me3 (Cat. No. C15410207). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left. </small></p>
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<div class="row">
<div class="small-12 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_IF.png" alt="H4K20me3 Antibody validated in Immunofluorescence" /></p>
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<div class="row">
<div class="small-12 columns">
<p><small><strong> Figure 6. Immunofluorescence using the Diagenode antibody directed against H4K20me3</strong><br /> HeLa cells were stained with the Diagenode antibody against H4K20me3 (Cat. No. C15410207) and with DAPI. Cells were fixed with methanol and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H4K20me3 antibody (left) diluted 1:500 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right. </small></p>
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'description' => 'Histones are the main constituents of the protein part of chromosomes of eukaryotic cells. They are rich in the amino acids arginine and lysine and have been greatly conserved during evolution. Histones pack the DNA into tight masses of chromatin. Two core histones of each class H2A, H2B, H3 and H4 assemble and are wrapped by 146 base pairs of DNA to form one octameric nucleosome. Histone tails undergo numerous post-translational modifications, which either directly or indirectly alter chromatin structure to facilitate transcriptional activation or repression or other nuclear processes. In addition to the genetic code, combinations of the different histone modifications reveal the so-called “histone code”. Histone methylation and demethylation is dynamically regulated by respectively histone methyl transferases and histone demethylases. Trimethylation of histone H4K20 is associated with inactive genomic regions, satellite repeats and ZNF gene repeats.',
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<th>Suggested dilution</th>
<th>References</th>
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<td>ChIP/ChIP-seq <sup>*</sup></td>
<td>0.5-1 µg per IP</td>
<td>Fig 1, 2</td>
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<tr>
<td>ELISA</td>
<td>1:500</td>
<td>Fig 3</td>
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<tr>
<td>Dot Blotting/Peptide array</td>
<td>1:20,000/1:10,000</td>
<td>Fig 4</td>
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<tr>
<td>Western Blotting</td>
<td>1:1,000</td>
<td>Fig 5</td>
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<tr>
<td>Immunofluorescence</td>
<td>1:500</td>
<td>Fig 6</td>
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<p><small><sup>*</sup> Please note that the optimal antibody amount per IP should be determined by the end-user. We recommend testing 0.5-5 µg per IP.</small></p>',
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'description' => '<p><span>Polyclonal antibody raised in rabbit against the region of histone H4 containing the trimethylated lysine 20 (H4K20me3), using a KLH-conjugated synthetic peptide. </span></p>',
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<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIP-a.png" alt="H4K20me3 Antibody ChIP Grade" /></p>
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<p><small><strong>Figure 1. ChIP results obtained with the Diagenode antibody directed against H4K20me3</strong><br />ChIP assays were performed using human HeLa cells, the Diagenode antibody against H4K20me3 (Cat. No. C15410207) and optimized PCR primer pairs for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (Cat. No. C01010051), using sheared chromatin from 1 million cells. The chromatin was spiked with a panel of in vitro assembled nucleosomes, each containing a specific lysine methylation (SNAP-ChIP K-MetStat Panel, Epicypher). A titration consisting of 0.2, 0.5, 1 and 2 µg of antibody per ChIP experiment was analyzed. IgG (1 µg/IP) was used as a negative IP control.<br /> <strong>Figure 1A.</strong> Quantitative PCR was performed with primers specific for the promoter of the active GAPDH and EIF4A2 genes, used as negative controls, and for the ZNF10 gene and the Sat2 satellite repeat, used as positive controls. The graph shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).<br /> <strong>Figure 1B.</strong> Recovery of the nucleosomes carrying the H4K20me1, H4K20me2, H4K2me3 and the unmodified H4K20 as determined by qPCR. The figure clearly shows the antibody is very specific in ChIP for the H4K20me3 modification. </small></p>
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<p>A. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-A.png" alt="H4K20me3 Antibody for ChIP assay" /></p>
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<div class="small-6 columns">
<p><small><strong> Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H4K20me3</strong><br /> ChIP was performed with 0.5 µg of the Diagenode antibody against H4K20me3 (Cat. No. C15410207) on sheared chromatin from 100,000 K562 cells using the “iDeal ChIP-seq” kit. The IP’d DNA was analysed by QPCR as described above (figure 2A). The IP’d DNA was subsequently analysed on an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. Figure 2B shows the signal distribution along the long arm of chromosome 19 and a zoomin to an enriched region containing several ZNF repeat genes. Figure 2C and D show the enrichment in the telomeric region of chromosome 12, also containing several ZNF repeat genes, and at ZNF510 on chromosome 9, respectively. The position of the amplicon used for ChIP-qPCR is indicated by an arrow. </small></p>
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<div class="row">
<div class="small-12 columns">
<p class="text-center">B. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-B.png" alt="H4K20me3 Antibody ChIP-seq Grade" /></p>
<p class="text-center">C. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-C.png" alt="H4K20me3 Antibody for ChIP-seq" /></p>
<p class="text-center">D. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-D.png" alt="H4K20me3 Antibody validated in ChIP-seq" /></p>
</div>
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<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ELISA.png" alt="H4K20me3 Antibody ELISA validation" /></p>
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<div class="small-6 columns">
<p><small><strong> Figure 3. Determination of the antibody titer</strong><br /> To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H4K20me3 (Cat. No. C15410207) in antigen coated wells. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the antibody was estimated to be 1:21,700. </small></p>
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<div class="row">
<div class="small-4 columns">
<p>A. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_CrossReactivity-A.png" alt="H4K20me3 Antibody Dot Blot validation" /></p>
<p>B. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_CrossReactivity-B.png" alt="H4K20me3 Antibody validated in Peptide Array " /></p>
</div>
<div class="small-8 columns">
<p><small><strong>Figure 4. Cross reactivity tests using the Diagenode antibody directed against H4K20me3</strong><br /> Figure 4A To test the cross reactivity of the Diagenode antibody against H4K20me3 (Cat. No. C15410207), a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H4K20. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 4A shows a high specificity of the antibody for the modification of interest. Figure 4B The specificity of the antibody was further demonstrated by peptide array analyses on an array containing 384 peptides with different combinations of modifications from histone H3, H4, H2A and H2B. The antibody was used at a dilution of 1:10,000. Figure 4B shows the specificity factor, calculated as the ratio of the average intensity of all spots containing the mark, divided by the average intensity of all spots not containing the mark. </small></p>
</div>
</div>
<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_WB.png" alt="H4K20me3 Antibody validated in Western Blot" /></p>
</div>
<div class="small-8 columns">
<p><small><strong> Figure 5. Western blot analysis using the Diagenode antibody directed against H4K20me3</strong><br /> Western blot was performed on whole cell (25 µg, lane 1) and histone extracts (15 µg, lane 2) from HeLa cells, and on 1 µg of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the Diagenode antibody against H4K20me3 (Cat. No. C15410207). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left. </small></p>
</div>
</div>
<div class="row">
<div class="small-12 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_IF.png" alt="H4K20me3 Antibody validated in Immunofluorescence" /></p>
</div>
</div>
<div class="row">
<div class="small-12 columns">
<p><small><strong> Figure 6. Immunofluorescence using the Diagenode antibody directed against H4K20me3</strong><br /> HeLa cells were stained with the Diagenode antibody against H4K20me3 (Cat. No. C15410207) and with DAPI. Cells were fixed with methanol and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H4K20me3 antibody (left) diluted 1:500 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right. </small></p>
</div>
</div>',
'label2' => 'Target Description',
'info2' => '<p>Histones are the main constituents of the protein part of chromosomes of eukaryotic cells. They are rich in the amino acids arginine and lysine and have been greatly conserved during evolution. Histones pack the DNA into tight masses of chromatin. Two core histones of each class H2A, H2B, H3 and H4 assemble and are wrapped by 146 base pairs of DNA to form one octameric nucleosome. Histone tails undergo numerous post-translational modifications, which either directly or indirectly alter chromatin structure to facilitate transcriptional activation or repression or other nuclear processes. In addition to the genetic code, combinations of the different histone modifications reveal the so-called “histone code”. Histone methylation and demethylation is dynamically regulated by respectively histone methyl transferases and histone demethylases. Trimethylation of histone H4K20 is associated with inactive genomic regions, satellite repeats and ZNF gene repeats.</p>',
'label3' => '',
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'format' => '50 μg',
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'old_catalog_number' => '',
'sf_code' => 'C15410207-D001-000581',
'type' => 'FRE',
'search_order' => '03-Antibody',
'price_EUR' => '480',
'price_USD' => '470',
'price_GBP' => '430',
'price_JPY' => '75190',
'price_CNY' => '',
'price_AUD' => '1175',
'country' => 'ALL',
'except_countries' => 'None',
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'online' => true,
'master' => true,
'last_datasheet_update' => 'December 19, 2019',
'slug' => 'h4k20me3-polyclonal-antibody-premium-50-mg-53-ml',
'meta_title' => 'H4K20me3 polyclonal antibody - Premium',
'meta_keywords' => '',
'meta_description' => 'H4K20me3 polyclonal antibody - Premium',
'modified' => '2024-01-17 16:52:00',
'created' => '2015-06-29 14:08:20',
'locale' => 'jpn'
),
'Antibody' => array(
'host' => '*****',
'id' => '80',
'name' => 'H4K20me3 polyclonal antibody',
'description' => 'Histones are the main constituents of the protein part of chromosomes of eukaryotic cells. They are rich in the amino acids arginine and lysine and have been greatly conserved during evolution. Histones pack the DNA into tight masses of chromatin. Two core histones of each class H2A, H2B, H3 and H4 assemble and are wrapped by 146 base pairs of DNA to form one octameric nucleosome. Histone tails undergo numerous post-translational modifications, which either directly or indirectly alter chromatin structure to facilitate transcriptional activation or repression or other nuclear processes. In addition to the genetic code, combinations of the different histone modifications reveal the so-called “histone code”. Histone methylation and demethylation is dynamically regulated by respectively histone methyl transferases and histone demethylases. Trimethylation of histone H4K20 is associated with inactive genomic regions, satellite repeats and ZNF gene repeats.',
'clonality' => '',
'isotype' => '',
'lot' => 'A2730P',
'concentration' => '0.94 µg/µl',
'reactivity' => 'Human, mouse, wide range expected',
'type' => 'Polyclonal',
'purity' => 'Affinity purified polyclonal antibody.',
'classification' => 'Premium',
'application_table' => '<table>
<thead>
<tr>
<th>Applications</th>
<th>Suggested dilution</th>
<th>References</th>
</tr>
</thead>
<tbody>
<tr>
<td>ChIP/ChIP-seq <sup>*</sup></td>
<td>0.5-1 µg per IP</td>
<td>Fig 1, 2</td>
</tr>
<tr>
<td>ELISA</td>
<td>1:500</td>
<td>Fig 3</td>
</tr>
<tr>
<td>Dot Blotting/Peptide array</td>
<td>1:20,000/1:10,000</td>
<td>Fig 4</td>
</tr>
<tr>
<td>Western Blotting</td>
<td>1:1,000</td>
<td>Fig 5</td>
</tr>
<tr>
<td>Immunofluorescence</td>
<td>1:500</td>
<td>Fig 6</td>
</tr>
</tbody>
</table>
<p></p>
<p><small><sup>*</sup> Please note that the optimal antibody amount per IP should be determined by the end-user. We recommend testing 0.5-5 µg per IP.</small></p>',
'storage_conditions' => 'Store at -20°C; for long storage, store at -80°C. Avoid multiple freeze-thaw cycles.',
'storage_buffer' => 'PBS containing 0.05% azide and 0.05% ProClin 300.',
'precautions' => 'This product is for research use only. Not for use in diagnostic or therapeutic procedures.',
'uniprot_acc' => '',
'slug' => '',
'meta_keywords' => '',
'meta_description' => '',
'modified' => '2020-02-25 13:33:22',
'created' => '0000-00-00 00:00:00',
'select_label' => '80 - H4K20me3 polyclonal antibody (A2730P - 0.94 µg/µl - Human, mouse, wide range expected - Affinity purified polyclonal antibody. - Rabbit)'
),
'Slave' => array(
(int) 0 => array(
'id' => '41',
'name' => 'C15410207',
'product_id' => '2284',
'modified' => '2016-02-18 20:47:29',
'created' => '2016-02-18 20:47:29'
)
),
'Group' => array(
'Group' => array(
'id' => '41',
'name' => 'C15410207',
'product_id' => '2284',
'modified' => '2016-02-18 20:47:29',
'created' => '2016-02-18 20:47:29'
),
'Master' => array(
'id' => '2284',
'antibody_id' => '80',
'name' => 'H4K20me3 Antibody',
'description' => '<p><span>Polyclonal antibody raised in rabbit against the region of histone <strong>H4 containing the trimethylated lysine 20 (H4K20me3)</strong>, using a KLH-conjugated synthetic peptide. </span></p>',
'label1' => 'Validation data',
'info1' => '<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIP-a.png" alt="H4K20me3 Antibody ChIP Grade" /></p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIP-b.png" alt="H4K20me3 Antibody for ChIP" /></p>
</div>
<div class="small-6 columns">
<p><small><strong>Figure 1. ChIP results obtained with the Diagenode antibody directed against H4K20me3</strong><br />ChIP assays were performed using human HeLa cells, the Diagenode antibody against H4K20me3 (Cat. No. C15410207) and optimized PCR primer pairs for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (Cat. No. C01010051), using sheared chromatin from 1 million cells. The chromatin was spiked with a panel of in vitro assembled nucleosomes, each containing a specific lysine methylation (SNAP-ChIP K-MetStat Panel, Epicypher). A titration consisting of 0.2, 0.5, 1 and 2 µg of antibody per ChIP experiment was analyzed. IgG (1 µg/IP) was used as a negative IP control.<br /> <strong>Figure 1A.</strong> Quantitative PCR was performed with primers specific for the promoter of the active GAPDH and EIF4A2 genes, used as negative controls, and for the ZNF10 gene and the Sat2 satellite repeat, used as positive controls. The graph shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).<br /> <strong>Figure 1B.</strong> Recovery of the nucleosomes carrying the H4K20me1, H4K20me2, H4K2me3 and the unmodified H4K20 as determined by qPCR. The figure clearly shows the antibody is very specific in ChIP for the H4K20me3 modification. </small></p>
</div>
</div>
<div class="extra-spaced"></div>
<div class="extra-spaced"></div>
<div class="extra-spaced"></div>
<div class="extra-spaced"></div>
<div class="extra-spaced"></div>
<div class="extra-spaced"></div>
<div class="extra-spaced"></div>
<div class="extra-spaced"></div>
<div class="extra-spaced"></div>
<div class="extra-spaced"></div>
<div class="extra-spaced"></div>
<div class="extra-spaced"></div>
<div class="extra-spaced"></div>
<div class="extra-spaced"></div>
<div class="extra-spaced"></div>
<div class="extra-spaced"></div>
<div class="extra-spaced"></div>
<div class="extra-spaced"></div>
<div class="extra-spaced"></div>
<div class="extra-spaced"></div>
<div class="row">
<div class="small-6 columns">
<p>A. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-A.png" alt="H4K20me3 Antibody for ChIP assay" /></p>
</div>
<div class="small-6 columns">
<p><small><strong> Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H4K20me3</strong><br /> ChIP was performed with 0.5 µg of the Diagenode antibody against H4K20me3 (Cat. No. C15410207) on sheared chromatin from 100,000 K562 cells using the “iDeal ChIP-seq” kit. The IP’d DNA was analysed by QPCR as described above (figure 2A). The IP’d DNA was subsequently analysed on an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. Figure 2B shows the signal distribution along the long arm of chromosome 19 and a zoomin to an enriched region containing several ZNF repeat genes. Figure 2C and D show the enrichment in the telomeric region of chromosome 12, also containing several ZNF repeat genes, and at ZNF510 on chromosome 9, respectively. The position of the amplicon used for ChIP-qPCR is indicated by an arrow. </small></p>
</div>
</div>
<div class="row">
<div class="small-12 columns">
<p class="text-center">B. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-B.png" alt="H4K20me3 Antibody ChIP-seq Grade" /></p>
<p class="text-center">C. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-C.png" alt="H4K20me3 Antibody for ChIP-seq" /></p>
<p class="text-center">D. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-D.png" alt="H4K20me3 Antibody validated in ChIP-seq" /></p>
</div>
</div>
<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ELISA.png" alt="H4K20me3 Antibody ELISA validation" /></p>
</div>
<div class="small-6 columns">
<p><small><strong> Figure 3. Determination of the antibody titer</strong><br /> To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H4K20me3 (Cat. No. C15410207) in antigen coated wells. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the antibody was estimated to be 1:21,700. </small></p>
</div>
</div>
<div class="row">
<div class="small-4 columns">
<p>A. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_CrossReactivity-A.png" alt="H4K20me3 Antibody Dot Blot validation" /></p>
<p>B. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_CrossReactivity-B.png" alt="H4K20me3 Antibody validated in Peptide Array " /></p>
</div>
<div class="small-8 columns">
<p><small><strong>Figure 4. Cross reactivity tests using the Diagenode antibody directed against H4K20me3</strong><br /> Figure 4A To test the cross reactivity of the Diagenode antibody against H4K20me3 (Cat. No. C15410207), a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H4K20. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 4A shows a high specificity of the antibody for the modification of interest. Figure 4B The specificity of the antibody was further demonstrated by peptide array analyses on an array containing 384 peptides with different combinations of modifications from histone H3, H4, H2A and H2B. The antibody was used at a dilution of 1:10,000. Figure 4B shows the specificity factor, calculated as the ratio of the average intensity of all spots containing the mark, divided by the average intensity of all spots not containing the mark. </small></p>
</div>
</div>
<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_WB.png" alt="H4K20me3 Antibody validated in Western Blot" /></p>
</div>
<div class="small-8 columns">
<p><small><strong> Figure 5. Western blot analysis using the Diagenode antibody directed against H4K20me3</strong><br /> Western blot was performed on whole cell (25 µg, lane 1) and histone extracts (15 µg, lane 2) from HeLa cells, and on 1 µg of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the Diagenode antibody against H4K20me3 (Cat. No. C15410207). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left. </small></p>
</div>
</div>
<div class="row">
<div class="small-12 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_IF.png" alt="H4K20me3 Antibody validated in Immunofluorescence" /></p>
</div>
</div>
<div class="row">
<div class="small-12 columns">
<p><small><strong> Figure 6. Immunofluorescence using the Diagenode antibody directed against H4K20me3</strong><br /> HeLa cells were stained with the Diagenode antibody against H4K20me3 (Cat. No. C15410207) and with DAPI. Cells were fixed with methanol and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H4K20me3 antibody (left) diluted 1:500 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right. </small></p>
</div>
</div>',
'label2' => 'Target Description',
'info2' => '<p>Histones are the main constituents of the protein part of chromosomes of eukaryotic cells. They are rich in the amino acids arginine and lysine and have been greatly conserved during evolution. Histones pack the DNA into tight masses of chromatin. Two core histones of each class H2A, H2B, H3 and H4 assemble and are wrapped by 146 base pairs of DNA to form one octameric nucleosome. Histone tails undergo numerous post-translational modifications, which either directly or indirectly alter chromatin structure to facilitate transcriptional activation or repression or other nuclear processes. In addition to the genetic code, combinations of the different histone modifications reveal the so-called “histone code”. Histone methylation and demethylation is dynamically regulated by respectively histone methyl transferases and histone demethylases. Trimethylation of histone H4K20 is associated with inactive genomic regions, satellite repeats and ZNF gene repeats.</p>',
'label3' => '',
'info3' => '',
'format' => '50 μg',
'catalog_number' => 'C15410207',
'old_catalog_number' => '',
'sf_code' => 'C15410207-D001-000581',
'type' => 'FRE',
'search_order' => '03-Antibody',
'price_EUR' => '480',
'price_USD' => '470',
'price_GBP' => '430',
'price_JPY' => '75190',
'price_CNY' => '',
'price_AUD' => '1175',
'country' => 'ALL',
'except_countries' => 'None',
'quote' => false,
'in_stock' => false,
'featured' => false,
'no_promo' => false,
'online' => true,
'master' => true,
'last_datasheet_update' => 'December 19, 2019',
'slug' => 'h4k20me3-polyclonal-antibody-premium-50-mg-53-ml',
'meta_title' => 'H4K20me3 Antibody - ChIP-seq Grade (C15410207) | Diagenode',
'meta_keywords' => '',
'meta_description' => 'H4K20me3 (Histone H4 trimethylated at lysine 20) Polyclonal Antibody validated in ChIP-seq, ChIP-qPCR, ELISA, DB, WB and IF. Specificity confirmed by Peptide array assay. Batch-specific data available on the website. Sample size available.',
'modified' => '2024-01-17 16:52:00',
'created' => '2015-06-29 14:08:20'
),
'Product' => array(
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[maximum depth reached]
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)
),
'Related' => array(
(int) 0 => array(
'id' => '2914',
'antibody_id' => null,
'name' => 'Auto ChIPmentation Kit for Histones - Replaced by C01011009',
'description' => '<div class="extra-spaced"><center></center></div>
<div class="row">
<div class="small-12 medium-8 large-8 columns"><!--<div class="extra-spaced"><center><a href="https://www.diagenode.com/en/pages/grant"><img src="https://www.diagenode.com/img/banners/banner-chipmentation-grant-580x120.jpg" /></a></center></div>-->
<div align="center"><video width="400" height="250" autoplay="autoplay" muted="" loop="loop" controls="controls" src="https://www.diagenode.com/videos/chipmentation-dgo.mp4" frameborder="0" allowfullscreen="allowfullscreen"></video></div>
<div align="center"><a href="https://www.diagenode.com/pages/form-chipmentation" class="center alert radius button"><i class="fa fa-info"></i> Contact us</a></div>
</div>
<div class="small-12 medium-4 large-4 columns"><center><a href="https://www.citeab.com/awards/2018/innovative-product-of-the-year" target="_blank"><img src="https://www.diagenode.com/emailing/images/citeab.png" width="224" height="200" /></a></center></div>
</div>
<p></p>
<p><strong>This product must be used with the <a href="https://www.diagenode.com/en/p/sx-8g-ip-star-compact-automated-system-1-unit">IP-Star Compact Automated System</a>.</strong></p>
<p>Diagenode’s latest technology for histone ChIP-seq, ChIPmentation, incorporates automation with an ligation-free protocol that integrates chromatin immunoprecipitation with NGS library preparation. Using histone ChIPmentation, you can avoid the multi-step ligation required for traditional histone ChIP-seq protocols, providing an easier protocol, faster time to results, and unmatched efficiency and reproducibility for histone ChIP-seq from challenging and low input samples. The ChIPmentaion Kit for Histones is a complete solution, including all buffers for chromatin preparation, immunoprecipitation, tagmentation and multiplexing using 24 single indexes.</p>',
'label1' => 'Characteristics',
'info1' => '<p>ChIPmentation is based on tagmentation that allows library preparation to be integrated during the ChIP itself using transposase and sequencing-compatible adaptors. ChIPmentation involves much easier and shorter protocol with high efficiency for low input samples using optimized ChIP and NGS sample preparation reagents on the IP-Star Compact Automated System. The IP-Star Compact Automated System ensures the highest reproducibility for challenging samples from limited input amounts.</p>
<h3>Benefits of the ChIPmentation system for histone ChIP-seq</h3>
<ul>
<li>Automates ChIP-seq of histones on the IP-Star® for highest reproducibility</li>
<li>Easier and faster than classical ChIP-seq</li>
<li>Validated for various histone marks</li>
<li>Ideal for analysis of large cohorts of samples</li>
<li>Ideal for analysis of large number of marks on a unique sample</li>
<li>High quality sequencing data</li>
</ul>
<p><img src="https://www.diagenode.com/img/product/chipmentation/chipmentation.jpg" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<h3>ChIPmentation - Highest reliability for even the lowest inputs</h3>
<p>The new Auto ChIPmentation technology has been tested on histone marks and compared to available datasets from the ENCODE project (Figure 1). ChIPmentation generated high quality data with low background. In addition, more than 99% of the top 40% peaks obtained with auto-ChIPmentation overlap with ENCODE datasets, which shows that ChIP-seq data obtained with ChIPmentation are highly reliable.</p>
<div class="spaced"><center>A.<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/auto-chipmentation.png" alt="Diagenode-Auto-ChIPmentation" /></center><br /><center>B. Match of the Top40 peaks of Auto-ChIPmentation<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/match-of-the-top40-peaks-of-auto-chipmentation.png" /></center></div>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure1"><i class="fa fa-caret-right"></i> Figure 1</a>
<div class="content" id="figure1">
<p><strong>Comparison of ChIPmentation sequencing results and reference ChIP-seq data.</strong><br /> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 100.000 cells was used for the immunoprecipitation with the Diagenode antibody targeting H3K4me3 (Cat. no. C15410003). The experiment was performed on the IP-Star® Compact Automated System. A. Distribution of the ChIPmentation and ENCODE readsets in a representative region of the genome. B. Comparison of the top 40% peaks from ChIPmentation dataset to the ENCODE dataset.</p>
</div>
</li>
</ul>
<p class="extra-spaced">As ChIPmentation is a ligation-free protocol and requires fewer steps than a classical workflow, it allows the generation of excellent ChIP-seq data from reduced starting amounts. The sequencing data generated from 5,000 and 10,000 cells are very similar to those obtained with 100,000 cells, showing more than 98% overlap of the top 40% peaks (Figure 2).</p>
<center>A.<br /><img class="extra-spaced" style="margin-bottom: 20px;" src="https://www.diagenode.com/img/categories/kits_chromatin_function/chipmentation-sequencing-figure-a.jpg" alt="Diagenode-ChIPmentation-sequencing" /></center>
<div class="row">
<div class="small-6 columns" style="text-align: left;">B. Match of the Top40 peaks of ChIPmentation from 10.000 cells<br /><center><img src="https://www.diagenode.com/img/categories/kits_chromatin_function/match-of-the-top40-peaks-of-chipmentation-from-cent-cells.png" /></center></div>
<div class="small-6 columns">C. Match of the Top40 peaks of ChIPmentation from 5.000 cells<center><img src="https://www.diagenode.com/img/categories/kits_chromatin_function/match-of-the-top40-peaks-of-chipmentation-from-five-cells.png" /></center></div>
</div>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure2"><i class="fa fa-caret-right"></i> Figure 2</a>
<div class="content" id="figure2">
<p><strong>ChIPmentation sequencing results obtained from decreasing starting amounts of cells.</strong><br /> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 100.000, 10.000 and 5.000 cells was used for the immunoprecipitation with the Diagenode antibody targeting H3K4me3 (Cat. no. C15410003). The experiments were performed on the IP-Star® Compact Automated System. A. Distribution of the ChIPmentation readsets in a representative region of the genome. B. and C. Comparison of the top 40% peaks from 10.000 (B.) and 5.000 (C.) cells with dataset generated from 100.000 cells.</p>
</div>
</li>
</ul>
<p class="extra-spaced">Moreover, in order to check the reproducibility of data generated with low cell amounts, correlation between replicates for 10.000 and 5.000 starting cells was performed (Figure 3). The data show very high correlations with Pearson’s coefficients of 0.96 for both conditions.</p>
<div class="row">
<div class="small-6 columns" style="text-align: left;"><center>A. Auto ChIPmentation on 10.000 cells<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/auto-chipmentation-on-10000cells.jpg" caption="false" width="480" height="420" /></center></div>
<div class="small-6 columns"><center>B. Auto ChIPmentation on 5.000 cells<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/auto-chipmentation-on-5000cells.jpg" width="480" height="420" /></center></div>
</div>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure3"><i class="fa fa-caret-right"></i> Figure 3</a>
<div class="content" id="figure3">
<p><strong>Correlation of replicates of auto-ChIPmentation experiments.</strong> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 10.000 and 5.000 cells was used for the immunoprecipitation with the Diagenode antibody targeting H3K4me3 (Cat. no. C15410003). The experiments were performed on the IP-Star® Compact Automated System.The scatterplots show the correlation between the enrichments between two replicates. The green line represents an ideal case where the two replicates are identical, while the red curve fitted on the data points by a LOESS regression shows the actual trend. A. Correlation analysis between two replicates of auto-ChIPmentation experiments starting from 10.000 cells. B. Correlation analysis between two replicates of auto-ChIPmentation experiments starting from 5.000 cells.</p>
</div>
</li>
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<p>The Auto-ChIPmentation workflow is easy and fast, producing high-quality ChIP-seq data even on low starting amounts.</p>
<center><img src="https://www.diagenode.com/img/product/chipmentation/chipmentation-sequencing-results-for-histone.jpg" width="678" height="240" /></center>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure4"><i class="fa fa-caret-right"></i> Figure 4</a>
<div class="content" id="figure4">
<p><strong>ChIPmentation sequencing results for four histone marks.</strong> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 1,000,000 cells was used for the immunoprecipitation with the Diagenode antibodies targeting H3K4me1 (Cat. no. C15410194), H3K27me3 (Cat. no. C15410195), H3K27ac (Cat. no. C15410196) and IgG (Cat. no. C15410206). The experiment was performed on the IP-Star® Compact Automated System.</p>
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<p><a href="https://www.diagenode.com/en/categories/chip-seq-grade-antibodies">ChIP-seq grade anti-histone antibodies</a> provide high yields with excellent specificity and sensitivity</p>',
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<p>Learn more about: <a href="https://www.diagenode.com/applications/western-blot">Loading control, MW marker visualization</a><em>. <br /></em></p>
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<p>Diagenode offers huge selection of highly sensitive antibodies validated in IF.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15200229-IF.jpg" alt="" height="245" width="256" /></p>
<p><sup><strong>Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong></sup></p>
<p><sup>HeLa cells transfected with a Cas9 expression vector (left) or untransfected cells (right) were fixed in methanol at -20°C, permeabilized with acetone at -20°C and blocked with PBS containing 2% BSA. The cells were stained with the Cas9 C-terminal antibody (Cat. No. C15200229) diluted 1:400, followed by incubation with an anti-mouse secondary antibody coupled to AF488. The bottom images show counter-staining of the nuclei with Hoechst 33342.</sup></p>
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'description' => '<p>Histones are the main protein components of chromatin involved in the compaction of DNA into nucleosomes, the basic units of chromatin. A <strong>nucleosome</strong> consists of one pair of each of the core histones (<strong>H2A</strong>, <strong>H2B</strong>, <strong>H3</strong> and <strong>H4</strong>) forming an octameric structure wrapped by 146 base pairs of DNA. The different nucleosomes are linked by the linker histone<strong> H1, </strong>allowing for further condensation of chromatin.</p>
<p>The core histones have a globular structure with large unstructured N-terminal tails protruding from the nucleosome. They can undergo to multiple post-translational modifications (PTM), mainly at the N-terminal tails. These <strong>post-translational modifications </strong>include methylation, acetylation, phosphorylation, ubiquitinylation, citrullination, sumoylation, deamination and crotonylation. The most well characterized PTMs are <strong>methylation,</strong> <strong>acetylation and phosphorylation</strong>. Histone methylation occurs mainly on lysine (K) residues, which can be mono-, di- or tri-methylated, and on arginines (R), which can be mono-methylated and symmetrically or asymmetrically di-methylated. Histone acetylation occurs on lysines and histone phosphorylation mainly on serines (S), threonines (T) and tyrosines (Y).</p>
<p>The PTMs of the different residues are involved in numerous processes such as DNA repair, DNA replication and chromosome condensation. They influence the chromatin organization and can be positively or negatively associated with gene expression. Trimethylation of H3K4, H3K36 and H3K79, and lysine acetylation generally result in an open chromatin configuration (figure below) and are therefore associated with <strong>euchromatin</strong> and gene activation. Trimethylation of H3K9, K3K27 and H4K20, on the other hand, is enriched in <strong>heterochromatin </strong>and associated with gene silencing. The combination of different histone modifications is called the "<strong>histone code</strong>”, analogous to the genetic code.</p>
<p><img src="https://www.diagenode.com/img/categories/antibodies/histone-marks-illustration.png" /></p>
<p>Diagenode is proud to offer a large range of antibodies against histones and histone modifications. Our antibodies are highly specific and have been validated in many applications, including <strong>ChIP</strong> and <strong>ChIP-seq</strong>.</p>
<p>Diagenode’s collection includes antibodies recognizing:</p>
<ul>
<li><strong>Histone H1 variants</strong></li>
<li><strong>Histone H2A, H2A variants and histone H2A</strong> <strong>modifications</strong> (serine phosphorylation, lysine acetylation, lysine ubiquitinylation)</li>
<li><strong>Histone H2B and H2B</strong> <strong>modifications </strong>(serine phosphorylation, lysine acetylation)</li>
<li><strong>Histone H3 and H3 modifications </strong>(lysine methylation (mono-, di- and tri-methylated), lysine acetylation, serine phosphorylation, threonine phosphorylation, arginine methylation (mono-methylated, symmetrically and asymmetrically di-methylated))</li>
<li><strong>Histone H4 and H4 modifications (</strong>lysine methylation (mono-, di- and tri-methylated), lysine acetylation, arginine methylation (mono-methylated and symmetrically di-methylated), serine phosphorylation )</li>
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<p><span style="font-weight: 400;"><strong>HDAC's HAT's, HMT's and other</strong> <strong>enzymes</strong> which modify histones can be found in the category <a href="../categories/chromatin-modifying-proteins-histone-transferase">Histone modifying enzymes</a><br /></span></p>
<p><span style="font-weight: 400;"> Diagenode’s highly validated antibodies:</span></p>
<ul>
<li><span style="font-weight: 400;"> Highly sensitive and specific</span></li>
<li><span style="font-weight: 400;"> Cost-effective (requires less antibody per reaction)</span></li>
<li><span style="font-weight: 400;"> Batch-specific data is available on the website</span></li>
<li><span style="font-weight: 400;"> Expert technical support</span></li>
<li><span style="font-weight: 400;"> Sample sizes available</span></li>
<li><span style="font-weight: 400;"> 100% satisfaction guarantee</span></li>
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<p><span style="font-weight: 400;">Diagenode provides leading solutions for epigenetic research. Because ChIP-seq is a widely-used technique, we validate our antibodies in ChIP and ChIP-seq experiments (in addition to conventional methods like Western blot, Dot blot, ELISA, and immunofluorescence) to provide the highest quality antibody. We standardize our validation and production to guarantee high product quality without technical bias. Diagenode guarantees ChIP-seq grade antibody performance under our suggested conditions.</span></p>
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<p><strong>ChIP-seq profile</strong> of active (H3K4me3 and H3K36me3) and inactive (H3K27me3) marks using Diagenode antibodies.</p>
<img src="https://www.diagenode.com/img/categories/antibodies/chip-seq-grade-antibodies.png" /></div>
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<p><small> ChIP was performed on sheared chromatin from 100,000 K562 cells using iDeal ChIP-seq kit for Histones (cat. No. C01010051) with 1 µg of the Diagenode antibodies against H3K27me3 (cat. No. C15410195) and H3K4me3 (cat. No. C15410003), and 0.5 µg of the antibody against H3K36me3 (cat. No. C15410192). The IP'd DNA was subsequently analysed on an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer's instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. The figure shows the signal distribution along the complete sequence of human chromosome 3, a zoomin to a 10 Mb region and a further zoomin to a 1.5 Mb region. </small></p>
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<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>
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<li>100% satisfaction guarantee</li>
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<p><span style="font-weight: 400;">Diagenode’s highly validated antibodies:</span></p>
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<div class="small-12 columns"><center></center>
<p><br />Chromatin immunoprecipitation (<b>ChIP</b>) is a technique to study the associations of proteins with the specific genomic regions in intact cells. One of the most important steps of this protocol is the immunoprecipitation of targeted protein using the antibody specifically recognizing it. The quality of antibodies used in ChIP is essential for the success of the experiment. Diagenode offers extensively validated ChIP-grade antibodies, confirmed for their specificity, and high level of performance in ChIP. Each batch is validated, and batch-specific data are available on the website.</p>
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<p><strong>ChIP results</strong> obtained with the antibody directed against H3K4me3 (Cat. No. <a href="../p/h3k4me3-polyclonal-antibody-premium-50-ug-50-ul">C15410003</a>). </p>
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<div class="small-12 medium-6 large-6 columns"><img src="https://www.diagenode.com/img/product/antibodies/C15410003-fig1-ChIP.jpg" alt="" width="400" height="315" /> </div>
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<p>Our aim at Diagenode is to offer the largest collection of highly specific <strong>ChIP-grade antibodies</strong>. We add new antibodies monthly. Find your ChIP-grade antibody in the list below and check more information about tested applications, extensive validation data, and product information.</p>',
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'name' => 'The 20S proteasome activator PA28γ controls the compaction of chromatin',
'authors' => 'Didier Fesquet, David Llères, Cristina Viganò, Francisca Méchali, Séverine Boulon, Robert Feil, Olivier Coux, Catherine Bonne-Andrea, Véronique Baldin',
'description' => '<p>The nuclear PA28γ is known to activate the 20S proteasome, but its precise cellular functions remains unclear. Here, we identify PA28γ as a key factor that structures heterochromatin. We find that in human cells, a fraction of PA28γ-20S proteasome complexes localizes within HP1-linked heterochromatin foci. Our biochemical studies show that PA28γ interacts with HP1 proteins, particularly HP1β, which recruits the PA28γ-20S proteasome complexes to heterochromatin. Loss of PA28γ does not modify the localization of HP1β, its mobility within nuclear foci, or the level of H3K9 tri-methylation, but reduces H4K20 mono- and tri-methylation, modifications involved in heterochromatin establishment. Concordantly, using a quantitative FRET-based microscopy assay to monitor nanometer-scale proximity between nucleosomes in living cells, we find that PA28γ regulates nucleosome proximity within heterochromatin, and thereby controls its compaction. This function of PA28γ is independent of the 20S proteasome. Importantly, HP1β on its own is unable to drive heterochromatin compaction without PA28γ. Combined, our data reveal an unexpected chromatin structural role of PA28γ, and provide new insights into the mechanism that controls HP1β-mediated heterochromatin compaction.</p>',
'date' => '2020-05-28',
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'authors' => 'Neagu A, van Genderen E, Escudero I, Verwegen L, Kurek D, Lehmann J, Stel J, Dirks RAM, van Mierlo G, Maas A, Eleveld C, Ge Y, den Dekker AT, Brouwer RWW, van IJcken WFJ, Modic M, Drukker M, Jansen JH, Rivron NC, Baart EB, Marks H, Ten Berge D',
'description' => '<p>Following implantation, the naive pluripotent epiblast of the mouse blastocyst generates a rosette, undergoes lumenogenesis and forms the primed pluripotent egg cylinder, which is able to generate the embryonic tissues. How pluripotency progression and morphogenesis are linked and whether intermediate pluripotent states exist remain controversial. We identify here a rosette pluripotent state defined by the co-expression of naive factors with the transcription factor OTX2. Downregulation of blastocyst WNT signals drives the transition into rosette pluripotency by inducing OTX2. The rosette then activates MEK signals that induce lumenogenesis and drive progression to primed pluripotency. Consequently, combined WNT and MEK inhibition supports rosette-like stem cells, a self-renewing naive-primed intermediate. Rosette-like stem cells erase constitutive heterochromatin marks and display a primed chromatin landscape, with bivalently marked primed pluripotency genes. Nonetheless, WNT induces reversion to naive pluripotency. The rosette is therefore a reversible pluripotent intermediate whereby control over both pluripotency progression and morphogenesis pivots from WNT to MEK signals.</p>',
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'description' => '<p>50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input. Single-molecule imaging shows that this step lowers transcription factor residence time and non-specific collisions during sampling for DNA targets. Third, chromatin interactions shift from long range to predominantly short range, and CTCF-mediated loops and contact domains double in numbers. This architectural change facilitates cognate promoter-enhancer contacts and also requires Myc and continual ATP production. Our results thus define the nature and transcriptional impact of chromatin decondensation and reveal an unexpected role for Myc in the establishment of nuclear topology in mammalian cells.</p>',
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<div class="row">
<div class="small-12 columns">
<a href="/jp/p/chipmentation-for-histones-24-rxns"><img src="/img/grey-logo.jpg" alt="default alt" class="th"/></a> </div>
<div class="small-12 columns">
<div class="small-6 columns" style="padding-left:0px;padding-right:0px;margin-top:-6px;margin-left:-1px">
<span class="success label" style="">C01011000</span>
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<!--a href="#" style="color:#B21329"><i class="fa fa-info-circle"></i></a-->
</div>
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<div class="small-12 columns" >
<h6 style="height:60px">Auto ChIPmentation Kit for Histones</h6>
</div>
</div>
</li>
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'description' => '<div class="extra-spaced"><center></center></div>
<div class="row">
<div class="small-12 medium-8 large-8 columns"><!--<div class="extra-spaced"><center><a href="https://www.diagenode.com/en/pages/grant"><img src="https://www.diagenode.com/img/banners/banner-chipmentation-grant-580x120.jpg" /></a></center></div>-->
<div align="center"><video width="400" height="250" autoplay="autoplay" muted="" loop="loop" controls="controls" src="https://www.diagenode.com/videos/chipmentation-dgo.mp4" frameborder="0" allowfullscreen="allowfullscreen"></video></div>
<div align="center"><a href="https://www.diagenode.com/pages/form-chipmentation" class="center alert radius button"><i class="fa fa-info"></i> Contact us</a></div>
</div>
<div class="small-12 medium-4 large-4 columns"><center><a href="https://www.citeab.com/awards/2018/innovative-product-of-the-year" target="_blank"><img src="https://www.diagenode.com/emailing/images/citeab.png" width="224" height="200" /></a></center></div>
</div>
<p></p>
<p><strong>This product must be used with the <a href="https://www.diagenode.com/en/p/sx-8g-ip-star-compact-automated-system-1-unit">IP-Star Compact Automated System</a>.</strong></p>
<p>Diagenode’s latest technology for histone ChIP-seq, ChIPmentation, incorporates automation with an ligation-free protocol that integrates chromatin immunoprecipitation with NGS library preparation. Using histone ChIPmentation, you can avoid the multi-step ligation required for traditional histone ChIP-seq protocols, providing an easier protocol, faster time to results, and unmatched efficiency and reproducibility for histone ChIP-seq from challenging and low input samples. The ChIPmentaion Kit for Histones is a complete solution, including all buffers for chromatin preparation, immunoprecipitation, tagmentation and multiplexing using 24 single indexes.</p>',
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'info1' => '<p>ChIPmentation is based on tagmentation that allows library preparation to be integrated during the ChIP itself using transposase and sequencing-compatible adaptors. ChIPmentation involves much easier and shorter protocol with high efficiency for low input samples using optimized ChIP and NGS sample preparation reagents on the IP-Star Compact Automated System. The IP-Star Compact Automated System ensures the highest reproducibility for challenging samples from limited input amounts.</p>
<h3>Benefits of the ChIPmentation system for histone ChIP-seq</h3>
<ul>
<li>Automates ChIP-seq of histones on the IP-Star® for highest reproducibility</li>
<li>Easier and faster than classical ChIP-seq</li>
<li>Validated for various histone marks</li>
<li>Ideal for analysis of large cohorts of samples</li>
<li>Ideal for analysis of large number of marks on a unique sample</li>
<li>High quality sequencing data</li>
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<p><img src="https://www.diagenode.com/img/product/chipmentation/chipmentation.jpg" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<h3>ChIPmentation - Highest reliability for even the lowest inputs</h3>
<p>The new Auto ChIPmentation technology has been tested on histone marks and compared to available datasets from the ENCODE project (Figure 1). ChIPmentation generated high quality data with low background. In addition, more than 99% of the top 40% peaks obtained with auto-ChIPmentation overlap with ENCODE datasets, which shows that ChIP-seq data obtained with ChIPmentation are highly reliable.</p>
<div class="spaced"><center>A.<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/auto-chipmentation.png" alt="Diagenode-Auto-ChIPmentation" /></center><br /><center>B. Match of the Top40 peaks of Auto-ChIPmentation<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/match-of-the-top40-peaks-of-auto-chipmentation.png" /></center></div>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure1"><i class="fa fa-caret-right"></i> Figure 1</a>
<div class="content" id="figure1">
<p><strong>Comparison of ChIPmentation sequencing results and reference ChIP-seq data.</strong><br /> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 100.000 cells was used for the immunoprecipitation with the Diagenode antibody targeting H3K4me3 (Cat. no. C15410003). The experiment was performed on the IP-Star® Compact Automated System. A. Distribution of the ChIPmentation and ENCODE readsets in a representative region of the genome. B. Comparison of the top 40% peaks from ChIPmentation dataset to the ENCODE dataset.</p>
</div>
</li>
</ul>
<p class="extra-spaced">As ChIPmentation is a ligation-free protocol and requires fewer steps than a classical workflow, it allows the generation of excellent ChIP-seq data from reduced starting amounts. The sequencing data generated from 5,000 and 10,000 cells are very similar to those obtained with 100,000 cells, showing more than 98% overlap of the top 40% peaks (Figure 2).</p>
<center>A.<br /><img class="extra-spaced" style="margin-bottom: 20px;" src="https://www.diagenode.com/img/categories/kits_chromatin_function/chipmentation-sequencing-figure-a.jpg" alt="Diagenode-ChIPmentation-sequencing" /></center>
<div class="row">
<div class="small-6 columns" style="text-align: left;">B. Match of the Top40 peaks of ChIPmentation from 10.000 cells<br /><center><img src="https://www.diagenode.com/img/categories/kits_chromatin_function/match-of-the-top40-peaks-of-chipmentation-from-cent-cells.png" /></center></div>
<div class="small-6 columns">C. Match of the Top40 peaks of ChIPmentation from 5.000 cells<center><img src="https://www.diagenode.com/img/categories/kits_chromatin_function/match-of-the-top40-peaks-of-chipmentation-from-five-cells.png" /></center></div>
</div>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure2"><i class="fa fa-caret-right"></i> Figure 2</a>
<div class="content" id="figure2">
<p><strong>ChIPmentation sequencing results obtained from decreasing starting amounts of cells.</strong><br /> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 100.000, 10.000 and 5.000 cells was used for the immunoprecipitation with the Diagenode antibody targeting H3K4me3 (Cat. no. C15410003). The experiments were performed on the IP-Star® Compact Automated System. A. Distribution of the ChIPmentation readsets in a representative region of the genome. B. and C. Comparison of the top 40% peaks from 10.000 (B.) and 5.000 (C.) cells with dataset generated from 100.000 cells.</p>
</div>
</li>
</ul>
<p class="extra-spaced">Moreover, in order to check the reproducibility of data generated with low cell amounts, correlation between replicates for 10.000 and 5.000 starting cells was performed (Figure 3). The data show very high correlations with Pearson’s coefficients of 0.96 for both conditions.</p>
<div class="row">
<div class="small-6 columns" style="text-align: left;"><center>A. Auto ChIPmentation on 10.000 cells<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/auto-chipmentation-on-10000cells.jpg" caption="false" width="480" height="420" /></center></div>
<div class="small-6 columns"><center>B. Auto ChIPmentation on 5.000 cells<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/auto-chipmentation-on-5000cells.jpg" width="480" height="420" /></center></div>
</div>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure3"><i class="fa fa-caret-right"></i> Figure 3</a>
<div class="content" id="figure3">
<p><strong>Correlation of replicates of auto-ChIPmentation experiments.</strong> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 10.000 and 5.000 cells was used for the immunoprecipitation with the Diagenode antibody targeting H3K4me3 (Cat. no. C15410003). The experiments were performed on the IP-Star® Compact Automated System.The scatterplots show the correlation between the enrichments between two replicates. The green line represents an ideal case where the two replicates are identical, while the red curve fitted on the data points by a LOESS regression shows the actual trend. A. Correlation analysis between two replicates of auto-ChIPmentation experiments starting from 10.000 cells. B. Correlation analysis between two replicates of auto-ChIPmentation experiments starting from 5.000 cells.</p>
</div>
</li>
</ul>
<p>The Auto-ChIPmentation workflow is easy and fast, producing high-quality ChIP-seq data even on low starting amounts.</p>
<center><img src="https://www.diagenode.com/img/product/chipmentation/chipmentation-sequencing-results-for-histone.jpg" width="678" height="240" /></center>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure4"><i class="fa fa-caret-right"></i> Figure 4</a>
<div class="content" id="figure4">
<p><strong>ChIPmentation sequencing results for four histone marks.</strong> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 1,000,000 cells was used for the immunoprecipitation with the Diagenode antibodies targeting H3K4me1 (Cat. no. C15410194), H3K27me3 (Cat. no. C15410195), H3K27ac (Cat. no. C15410196) and IgG (Cat. no. C15410206). The experiment was performed on the IP-Star® Compact Automated System.</p>
</div>
</li>
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<p><a href="https://www.diagenode.com/en/categories/chip-seq-grade-antibodies">ChIP-seq grade anti-histone antibodies</a> provide high yields with excellent specificity and sensitivity</p>',
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIP-a.png" alt="H4K20me3 Antibody ChIP Grade" /></p>
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<p><small><strong>Figure 1. ChIP results obtained with the Diagenode antibody directed against H4K20me3</strong><br />ChIP assays were performed using human HeLa cells, the Diagenode antibody against H4K20me3 (Cat. No. C15410207) and optimized PCR primer pairs for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (Cat. No. C01010051), using sheared chromatin from 1 million cells. The chromatin was spiked with a panel of in vitro assembled nucleosomes, each containing a specific lysine methylation (SNAP-ChIP K-MetStat Panel, Epicypher). A titration consisting of 0.2, 0.5, 1 and 2 µg of antibody per ChIP experiment was analyzed. IgG (1 µg/IP) was used as a negative IP control.<br /> <strong>Figure 1A.</strong> Quantitative PCR was performed with primers specific for the promoter of the active GAPDH and EIF4A2 genes, used as negative controls, and for the ZNF10 gene and the Sat2 satellite repeat, used as positive controls. The graph shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).<br /> <strong>Figure 1B.</strong> Recovery of the nucleosomes carrying the H4K20me1, H4K20me2, H4K2me3 and the unmodified H4K20 as determined by qPCR. The figure clearly shows the antibody is very specific in ChIP for the H4K20me3 modification. </small></p>
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<p>A. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-A.png" alt="H4K20me3 Antibody for ChIP assay" /></p>
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<div class="small-6 columns">
<p><small><strong> Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H4K20me3</strong><br /> ChIP was performed with 0.5 µg of the Diagenode antibody against H4K20me3 (Cat. No. C15410207) on sheared chromatin from 100,000 K562 cells using the “iDeal ChIP-seq” kit. The IP’d DNA was analysed by QPCR as described above (figure 2A). The IP’d DNA was subsequently analysed on an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. Figure 2B shows the signal distribution along the long arm of chromosome 19 and a zoomin to an enriched region containing several ZNF repeat genes. Figure 2C and D show the enrichment in the telomeric region of chromosome 12, also containing several ZNF repeat genes, and at ZNF510 on chromosome 9, respectively. The position of the amplicon used for ChIP-qPCR is indicated by an arrow. </small></p>
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<p class="text-center">B. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-B.png" alt="H4K20me3 Antibody ChIP-seq Grade" /></p>
<p class="text-center">C. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-C.png" alt="H4K20me3 Antibody for ChIP-seq" /></p>
<p class="text-center">D. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-D.png" alt="H4K20me3 Antibody validated in ChIP-seq" /></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ELISA.png" alt="H4K20me3 Antibody ELISA validation" /></p>
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<p><small><strong> Figure 3. Determination of the antibody titer</strong><br /> To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H4K20me3 (Cat. No. C15410207) in antigen coated wells. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the antibody was estimated to be 1:21,700. </small></p>
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<p>A. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_CrossReactivity-A.png" alt="H4K20me3 Antibody Dot Blot validation" /></p>
<p>B. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_CrossReactivity-B.png" alt="H4K20me3 Antibody validated in Peptide Array " /></p>
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<p><small><strong>Figure 4. Cross reactivity tests using the Diagenode antibody directed against H4K20me3</strong><br /> Figure 4A To test the cross reactivity of the Diagenode antibody against H4K20me3 (Cat. No. C15410207), a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H4K20. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 4A shows a high specificity of the antibody for the modification of interest. Figure 4B The specificity of the antibody was further demonstrated by peptide array analyses on an array containing 384 peptides with different combinations of modifications from histone H3, H4, H2A and H2B. The antibody was used at a dilution of 1:10,000. Figure 4B shows the specificity factor, calculated as the ratio of the average intensity of all spots containing the mark, divided by the average intensity of all spots not containing the mark. </small></p>
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<p><small><strong> Figure 5. Western blot analysis using the Diagenode antibody directed against H4K20me3</strong><br /> Western blot was performed on whole cell (25 µg, lane 1) and histone extracts (15 µg, lane 2) from HeLa cells, and on 1 µg of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the Diagenode antibody against H4K20me3 (Cat. No. C15410207). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left. </small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_IF.png" alt="H4K20me3 Antibody validated in Immunofluorescence" /></p>
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<p><small><strong> Figure 6. Immunofluorescence using the Diagenode antibody directed against H4K20me3</strong><br /> HeLa cells were stained with the Diagenode antibody against H4K20me3 (Cat. No. C15410207) and with DAPI. Cells were fixed with methanol and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H4K20me3 antibody (left) diluted 1:500 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right. </small></p>
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<p><small><strong>Figure 1. ChIP results obtained with the Diagenode antibody directed against H4K20me3</strong><br />ChIP assays were performed using human HeLa cells, the Diagenode antibody against H4K20me3 (Cat. No. C15410207) and optimized PCR primer pairs for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (Cat. No. C01010051), using sheared chromatin from 1 million cells. The chromatin was spiked with a panel of in vitro assembled nucleosomes, each containing a specific lysine methylation (SNAP-ChIP K-MetStat Panel, Epicypher). A titration consisting of 0.2, 0.5, 1 and 2 µg of antibody per ChIP experiment was analyzed. IgG (1 µg/IP) was used as a negative IP control.<br /> <strong>Figure 1A.</strong> Quantitative PCR was performed with primers specific for the promoter of the active GAPDH and EIF4A2 genes, used as negative controls, and for the ZNF10 gene and the Sat2 satellite repeat, used as positive controls. The graph shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).<br /> <strong>Figure 1B.</strong> Recovery of the nucleosomes carrying the H4K20me1, H4K20me2, H4K2me3 and the unmodified H4K20 as determined by qPCR. The figure clearly shows the antibody is very specific in ChIP for the H4K20me3 modification. </small></p>
</div>
</div>
<div class="extra-spaced"></div>
<div class="extra-spaced"></div>
<div class="extra-spaced"></div>
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<div class="row">
<div class="small-6 columns">
<p>A. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-A.png" alt="H4K20me3 Antibody for ChIP assay" /></p>
</div>
<div class="small-6 columns">
<p><small><strong> Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H4K20me3</strong><br /> ChIP was performed with 0.5 µg of the Diagenode antibody against H4K20me3 (Cat. No. C15410207) on sheared chromatin from 100,000 K562 cells using the “iDeal ChIP-seq” kit. The IP’d DNA was analysed by QPCR as described above (figure 2A). The IP’d DNA was subsequently analysed on an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. Figure 2B shows the signal distribution along the long arm of chromosome 19 and a zoomin to an enriched region containing several ZNF repeat genes. Figure 2C and D show the enrichment in the telomeric region of chromosome 12, also containing several ZNF repeat genes, and at ZNF510 on chromosome 9, respectively. The position of the amplicon used for ChIP-qPCR is indicated by an arrow. </small></p>
</div>
</div>
<div class="row">
<div class="small-12 columns">
<p class="text-center">B. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-B.png" alt="H4K20me3 Antibody ChIP-seq Grade" /></p>
<p class="text-center">C. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-C.png" alt="H4K20me3 Antibody for ChIP-seq" /></p>
<p class="text-center">D. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-D.png" alt="H4K20me3 Antibody validated in ChIP-seq" /></p>
</div>
</div>
<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ELISA.png" alt="H4K20me3 Antibody ELISA validation" /></p>
</div>
<div class="small-6 columns">
<p><small><strong> Figure 3. Determination of the antibody titer</strong><br /> To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H4K20me3 (Cat. No. C15410207) in antigen coated wells. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the antibody was estimated to be 1:21,700. </small></p>
</div>
</div>
<div class="row">
<div class="small-4 columns">
<p>A. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_CrossReactivity-A.png" alt="H4K20me3 Antibody Dot Blot validation" /></p>
<p>B. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_CrossReactivity-B.png" alt="H4K20me3 Antibody validated in Peptide Array " /></p>
</div>
<div class="small-8 columns">
<p><small><strong>Figure 4. Cross reactivity tests using the Diagenode antibody directed against H4K20me3</strong><br /> Figure 4A To test the cross reactivity of the Diagenode antibody against H4K20me3 (Cat. No. C15410207), a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H4K20. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 4A shows a high specificity of the antibody for the modification of interest. Figure 4B The specificity of the antibody was further demonstrated by peptide array analyses on an array containing 384 peptides with different combinations of modifications from histone H3, H4, H2A and H2B. The antibody was used at a dilution of 1:10,000. Figure 4B shows the specificity factor, calculated as the ratio of the average intensity of all spots containing the mark, divided by the average intensity of all spots not containing the mark. </small></p>
</div>
</div>
<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_WB.png" alt="H4K20me3 Antibody validated in Western Blot" /></p>
</div>
<div class="small-8 columns">
<p><small><strong> Figure 5. Western blot analysis using the Diagenode antibody directed against H4K20me3</strong><br /> Western blot was performed on whole cell (25 µg, lane 1) and histone extracts (15 µg, lane 2) from HeLa cells, and on 1 µg of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the Diagenode antibody against H4K20me3 (Cat. No. C15410207). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left. </small></p>
</div>
</div>
<div class="row">
<div class="small-12 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_IF.png" alt="H4K20me3 Antibody validated in Immunofluorescence" /></p>
</div>
</div>
<div class="row">
<div class="small-12 columns">
<p><small><strong> Figure 6. Immunofluorescence using the Diagenode antibody directed against H4K20me3</strong><br /> HeLa cells were stained with the Diagenode antibody against H4K20me3 (Cat. No. C15410207) and with DAPI. Cells were fixed with methanol and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H4K20me3 antibody (left) diluted 1:500 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right. </small></p>
</div>
</div>',
'label2' => 'Target Description',
'info2' => '<p>Histones are the main constituents of the protein part of chromosomes of eukaryotic cells. They are rich in the amino acids arginine and lysine and have been greatly conserved during evolution. Histones pack the DNA into tight masses of chromatin. Two core histones of each class H2A, H2B, H3 and H4 assemble and are wrapped by 146 base pairs of DNA to form one octameric nucleosome. Histone tails undergo numerous post-translational modifications, which either directly or indirectly alter chromatin structure to facilitate transcriptional activation or repression or other nuclear processes. In addition to the genetic code, combinations of the different histone modifications reveal the so-called “histone code”. Histone methylation and demethylation is dynamically regulated by respectively histone methyl transferases and histone demethylases. Trimethylation of histone H4K20 is associated with inactive genomic regions, satellite repeats and ZNF gene repeats.</p>',
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'price_GBP' => '430',
'price_JPY' => '75190',
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'slug' => 'h4k20me3-polyclonal-antibody-premium-50-mg-53-ml',
'meta_title' => 'H4K20me3 polyclonal antibody - Premium',
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'meta_description' => 'H4K20me3 polyclonal antibody - Premium',
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'created' => '2015-06-29 14:08:20',
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),
'Antibody' => array(
'host' => '*****',
'id' => '80',
'name' => 'H4K20me3 polyclonal antibody',
'description' => 'Histones are the main constituents of the protein part of chromosomes of eukaryotic cells. They are rich in the amino acids arginine and lysine and have been greatly conserved during evolution. Histones pack the DNA into tight masses of chromatin. Two core histones of each class H2A, H2B, H3 and H4 assemble and are wrapped by 146 base pairs of DNA to form one octameric nucleosome. Histone tails undergo numerous post-translational modifications, which either directly or indirectly alter chromatin structure to facilitate transcriptional activation or repression or other nuclear processes. In addition to the genetic code, combinations of the different histone modifications reveal the so-called “histone code”. Histone methylation and demethylation is dynamically regulated by respectively histone methyl transferases and histone demethylases. Trimethylation of histone H4K20 is associated with inactive genomic regions, satellite repeats and ZNF gene repeats.',
'clonality' => '',
'isotype' => '',
'lot' => 'A2730P',
'concentration' => '0.94 µg/µl',
'reactivity' => 'Human, mouse, wide range expected',
'type' => 'Polyclonal',
'purity' => 'Affinity purified polyclonal antibody.',
'classification' => 'Premium',
'application_table' => '<table>
<thead>
<tr>
<th>Applications</th>
<th>Suggested dilution</th>
<th>References</th>
</tr>
</thead>
<tbody>
<tr>
<td>ChIP/ChIP-seq <sup>*</sup></td>
<td>0.5-1 µg per IP</td>
<td>Fig 1, 2</td>
</tr>
<tr>
<td>ELISA</td>
<td>1:500</td>
<td>Fig 3</td>
</tr>
<tr>
<td>Dot Blotting/Peptide array</td>
<td>1:20,000/1:10,000</td>
<td>Fig 4</td>
</tr>
<tr>
<td>Western Blotting</td>
<td>1:1,000</td>
<td>Fig 5</td>
</tr>
<tr>
<td>Immunofluorescence</td>
<td>1:500</td>
<td>Fig 6</td>
</tr>
</tbody>
</table>
<p></p>
<p><small><sup>*</sup> Please note that the optimal antibody amount per IP should be determined by the end-user. We recommend testing 0.5-5 µg per IP.</small></p>',
'storage_conditions' => 'Store at -20°C; for long storage, store at -80°C. Avoid multiple freeze-thaw cycles.',
'storage_buffer' => 'PBS containing 0.05% azide and 0.05% ProClin 300.',
'precautions' => 'This product is for research use only. Not for use in diagnostic or therapeutic procedures.',
'uniprot_acc' => '',
'slug' => '',
'meta_keywords' => '',
'meta_description' => '',
'modified' => '2020-02-25 13:33:22',
'created' => '0000-00-00 00:00:00',
'select_label' => '80 - H4K20me3 polyclonal antibody (A2730P - 0.94 µg/µl - Human, mouse, wide range expected - Affinity purified polyclonal antibody. - Rabbit)'
),
'Slave' => array(
(int) 0 => array(
'id' => '41',
'name' => 'C15410207',
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'modified' => '2016-02-18 20:47:29',
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),
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'Group' => array(
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),
'Master' => array(
'id' => '2284',
'antibody_id' => '80',
'name' => 'H4K20me3 Antibody',
'description' => '<p><span>Polyclonal antibody raised in rabbit against the region of histone <strong>H4 containing the trimethylated lysine 20 (H4K20me3)</strong>, using a KLH-conjugated synthetic peptide. </span></p>',
'label1' => 'Validation data',
'info1' => '<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIP-a.png" alt="H4K20me3 Antibody ChIP Grade" /></p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIP-b.png" alt="H4K20me3 Antibody for ChIP" /></p>
</div>
<div class="small-6 columns">
<p><small><strong>Figure 1. ChIP results obtained with the Diagenode antibody directed against H4K20me3</strong><br />ChIP assays were performed using human HeLa cells, the Diagenode antibody against H4K20me3 (Cat. No. C15410207) and optimized PCR primer pairs for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (Cat. No. C01010051), using sheared chromatin from 1 million cells. The chromatin was spiked with a panel of in vitro assembled nucleosomes, each containing a specific lysine methylation (SNAP-ChIP K-MetStat Panel, Epicypher). A titration consisting of 0.2, 0.5, 1 and 2 µg of antibody per ChIP experiment was analyzed. IgG (1 µg/IP) was used as a negative IP control.<br /> <strong>Figure 1A.</strong> Quantitative PCR was performed with primers specific for the promoter of the active GAPDH and EIF4A2 genes, used as negative controls, and for the ZNF10 gene and the Sat2 satellite repeat, used as positive controls. The graph shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).<br /> <strong>Figure 1B.</strong> Recovery of the nucleosomes carrying the H4K20me1, H4K20me2, H4K2me3 and the unmodified H4K20 as determined by qPCR. The figure clearly shows the antibody is very specific in ChIP for the H4K20me3 modification. </small></p>
</div>
</div>
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<div class="extra-spaced"></div>
<div class="extra-spaced"></div>
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<div class="row">
<div class="small-6 columns">
<p>A. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-A.png" alt="H4K20me3 Antibody for ChIP assay" /></p>
</div>
<div class="small-6 columns">
<p><small><strong> Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H4K20me3</strong><br /> ChIP was performed with 0.5 µg of the Diagenode antibody against H4K20me3 (Cat. No. C15410207) on sheared chromatin from 100,000 K562 cells using the “iDeal ChIP-seq” kit. The IP’d DNA was analysed by QPCR as described above (figure 2A). The IP’d DNA was subsequently analysed on an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. Figure 2B shows the signal distribution along the long arm of chromosome 19 and a zoomin to an enriched region containing several ZNF repeat genes. Figure 2C and D show the enrichment in the telomeric region of chromosome 12, also containing several ZNF repeat genes, and at ZNF510 on chromosome 9, respectively. The position of the amplicon used for ChIP-qPCR is indicated by an arrow. </small></p>
</div>
</div>
<div class="row">
<div class="small-12 columns">
<p class="text-center">B. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-B.png" alt="H4K20me3 Antibody ChIP-seq Grade" /></p>
<p class="text-center">C. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-C.png" alt="H4K20me3 Antibody for ChIP-seq" /></p>
<p class="text-center">D. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-D.png" alt="H4K20me3 Antibody validated in ChIP-seq" /></p>
</div>
</div>
<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ELISA.png" alt="H4K20me3 Antibody ELISA validation" /></p>
</div>
<div class="small-6 columns">
<p><small><strong> Figure 3. Determination of the antibody titer</strong><br /> To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H4K20me3 (Cat. No. C15410207) in antigen coated wells. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the antibody was estimated to be 1:21,700. </small></p>
</div>
</div>
<div class="row">
<div class="small-4 columns">
<p>A. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_CrossReactivity-A.png" alt="H4K20me3 Antibody Dot Blot validation" /></p>
<p>B. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_CrossReactivity-B.png" alt="H4K20me3 Antibody validated in Peptide Array " /></p>
</div>
<div class="small-8 columns">
<p><small><strong>Figure 4. Cross reactivity tests using the Diagenode antibody directed against H4K20me3</strong><br /> Figure 4A To test the cross reactivity of the Diagenode antibody against H4K20me3 (Cat. No. C15410207), a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H4K20. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 4A shows a high specificity of the antibody for the modification of interest. Figure 4B The specificity of the antibody was further demonstrated by peptide array analyses on an array containing 384 peptides with different combinations of modifications from histone H3, H4, H2A and H2B. The antibody was used at a dilution of 1:10,000. Figure 4B shows the specificity factor, calculated as the ratio of the average intensity of all spots containing the mark, divided by the average intensity of all spots not containing the mark. </small></p>
</div>
</div>
<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_WB.png" alt="H4K20me3 Antibody validated in Western Blot" /></p>
</div>
<div class="small-8 columns">
<p><small><strong> Figure 5. Western blot analysis using the Diagenode antibody directed against H4K20me3</strong><br /> Western blot was performed on whole cell (25 µg, lane 1) and histone extracts (15 µg, lane 2) from HeLa cells, and on 1 µg of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the Diagenode antibody against H4K20me3 (Cat. No. C15410207). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left. </small></p>
</div>
</div>
<div class="row">
<div class="small-12 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_IF.png" alt="H4K20me3 Antibody validated in Immunofluorescence" /></p>
</div>
</div>
<div class="row">
<div class="small-12 columns">
<p><small><strong> Figure 6. Immunofluorescence using the Diagenode antibody directed against H4K20me3</strong><br /> HeLa cells were stained with the Diagenode antibody against H4K20me3 (Cat. No. C15410207) and with DAPI. Cells were fixed with methanol and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H4K20me3 antibody (left) diluted 1:500 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right. </small></p>
</div>
</div>',
'label2' => 'Target Description',
'info2' => '<p>Histones are the main constituents of the protein part of chromosomes of eukaryotic cells. They are rich in the amino acids arginine and lysine and have been greatly conserved during evolution. Histones pack the DNA into tight masses of chromatin. Two core histones of each class H2A, H2B, H3 and H4 assemble and are wrapped by 146 base pairs of DNA to form one octameric nucleosome. Histone tails undergo numerous post-translational modifications, which either directly or indirectly alter chromatin structure to facilitate transcriptional activation or repression or other nuclear processes. In addition to the genetic code, combinations of the different histone modifications reveal the so-called “histone code”. Histone methylation and demethylation is dynamically regulated by respectively histone methyl transferases and histone demethylases. Trimethylation of histone H4K20 is associated with inactive genomic regions, satellite repeats and ZNF gene repeats.</p>',
'label3' => '',
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'format' => '50 μg',
'catalog_number' => 'C15410207',
'old_catalog_number' => '',
'sf_code' => 'C15410207-D001-000581',
'type' => 'FRE',
'search_order' => '03-Antibody',
'price_EUR' => '480',
'price_USD' => '470',
'price_GBP' => '430',
'price_JPY' => '75190',
'price_CNY' => '',
'price_AUD' => '1175',
'country' => 'ALL',
'except_countries' => 'None',
'quote' => false,
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'online' => true,
'master' => true,
'last_datasheet_update' => 'December 19, 2019',
'slug' => 'h4k20me3-polyclonal-antibody-premium-50-mg-53-ml',
'meta_title' => 'H4K20me3 Antibody - ChIP-seq Grade (C15410207) | Diagenode',
'meta_keywords' => '',
'meta_description' => 'H4K20me3 (Histone H4 trimethylated at lysine 20) Polyclonal Antibody validated in ChIP-seq, ChIP-qPCR, ELISA, DB, WB and IF. Specificity confirmed by Peptide array assay. Batch-specific data available on the website. Sample size available.',
'modified' => '2024-01-17 16:52:00',
'created' => '2015-06-29 14:08:20'
),
'Product' => array(
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'Related' => array(
(int) 0 => array(
'id' => '2914',
'antibody_id' => null,
'name' => 'Auto ChIPmentation Kit for Histones - Replaced by C01011009',
'description' => '<div class="extra-spaced"><center></center></div>
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<p></p>
<p><strong>This product must be used with the <a href="https://www.diagenode.com/en/p/sx-8g-ip-star-compact-automated-system-1-unit">IP-Star Compact Automated System</a>.</strong></p>
<p>Diagenode’s latest technology for histone ChIP-seq, ChIPmentation, incorporates automation with an ligation-free protocol that integrates chromatin immunoprecipitation with NGS library preparation. Using histone ChIPmentation, you can avoid the multi-step ligation required for traditional histone ChIP-seq protocols, providing an easier protocol, faster time to results, and unmatched efficiency and reproducibility for histone ChIP-seq from challenging and low input samples. The ChIPmentaion Kit for Histones is a complete solution, including all buffers for chromatin preparation, immunoprecipitation, tagmentation and multiplexing using 24 single indexes.</p>',
'label1' => 'Characteristics',
'info1' => '<p>ChIPmentation is based on tagmentation that allows library preparation to be integrated during the ChIP itself using transposase and sequencing-compatible adaptors. ChIPmentation involves much easier and shorter protocol with high efficiency for low input samples using optimized ChIP and NGS sample preparation reagents on the IP-Star Compact Automated System. The IP-Star Compact Automated System ensures the highest reproducibility for challenging samples from limited input amounts.</p>
<h3>Benefits of the ChIPmentation system for histone ChIP-seq</h3>
<ul>
<li>Automates ChIP-seq of histones on the IP-Star® for highest reproducibility</li>
<li>Easier and faster than classical ChIP-seq</li>
<li>Validated for various histone marks</li>
<li>Ideal for analysis of large cohorts of samples</li>
<li>Ideal for analysis of large number of marks on a unique sample</li>
<li>High quality sequencing data</li>
</ul>
<p><img src="https://www.diagenode.com/img/product/chipmentation/chipmentation.jpg" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<h3>ChIPmentation - Highest reliability for even the lowest inputs</h3>
<p>The new Auto ChIPmentation technology has been tested on histone marks and compared to available datasets from the ENCODE project (Figure 1). ChIPmentation generated high quality data with low background. In addition, more than 99% of the top 40% peaks obtained with auto-ChIPmentation overlap with ENCODE datasets, which shows that ChIP-seq data obtained with ChIPmentation are highly reliable.</p>
<div class="spaced"><center>A.<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/auto-chipmentation.png" alt="Diagenode-Auto-ChIPmentation" /></center><br /><center>B. Match of the Top40 peaks of Auto-ChIPmentation<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/match-of-the-top40-peaks-of-auto-chipmentation.png" /></center></div>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure1"><i class="fa fa-caret-right"></i> Figure 1</a>
<div class="content" id="figure1">
<p><strong>Comparison of ChIPmentation sequencing results and reference ChIP-seq data.</strong><br /> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 100.000 cells was used for the immunoprecipitation with the Diagenode antibody targeting H3K4me3 (Cat. no. C15410003). The experiment was performed on the IP-Star® Compact Automated System. A. Distribution of the ChIPmentation and ENCODE readsets in a representative region of the genome. B. Comparison of the top 40% peaks from ChIPmentation dataset to the ENCODE dataset.</p>
</div>
</li>
</ul>
<p class="extra-spaced">As ChIPmentation is a ligation-free protocol and requires fewer steps than a classical workflow, it allows the generation of excellent ChIP-seq data from reduced starting amounts. The sequencing data generated from 5,000 and 10,000 cells are very similar to those obtained with 100,000 cells, showing more than 98% overlap of the top 40% peaks (Figure 2).</p>
<center>A.<br /><img class="extra-spaced" style="margin-bottom: 20px;" src="https://www.diagenode.com/img/categories/kits_chromatin_function/chipmentation-sequencing-figure-a.jpg" alt="Diagenode-ChIPmentation-sequencing" /></center>
<div class="row">
<div class="small-6 columns" style="text-align: left;">B. Match of the Top40 peaks of ChIPmentation from 10.000 cells<br /><center><img src="https://www.diagenode.com/img/categories/kits_chromatin_function/match-of-the-top40-peaks-of-chipmentation-from-cent-cells.png" /></center></div>
<div class="small-6 columns">C. Match of the Top40 peaks of ChIPmentation from 5.000 cells<center><img src="https://www.diagenode.com/img/categories/kits_chromatin_function/match-of-the-top40-peaks-of-chipmentation-from-five-cells.png" /></center></div>
</div>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure2"><i class="fa fa-caret-right"></i> Figure 2</a>
<div class="content" id="figure2">
<p><strong>ChIPmentation sequencing results obtained from decreasing starting amounts of cells.</strong><br /> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 100.000, 10.000 and 5.000 cells was used for the immunoprecipitation with the Diagenode antibody targeting H3K4me3 (Cat. no. C15410003). The experiments were performed on the IP-Star® Compact Automated System. A. Distribution of the ChIPmentation readsets in a representative region of the genome. B. and C. Comparison of the top 40% peaks from 10.000 (B.) and 5.000 (C.) cells with dataset generated from 100.000 cells.</p>
</div>
</li>
</ul>
<p class="extra-spaced">Moreover, in order to check the reproducibility of data generated with low cell amounts, correlation between replicates for 10.000 and 5.000 starting cells was performed (Figure 3). The data show very high correlations with Pearson’s coefficients of 0.96 for both conditions.</p>
<div class="row">
<div class="small-6 columns" style="text-align: left;"><center>A. Auto ChIPmentation on 10.000 cells<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/auto-chipmentation-on-10000cells.jpg" caption="false" width="480" height="420" /></center></div>
<div class="small-6 columns"><center>B. Auto ChIPmentation on 5.000 cells<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/auto-chipmentation-on-5000cells.jpg" width="480" height="420" /></center></div>
</div>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure3"><i class="fa fa-caret-right"></i> Figure 3</a>
<div class="content" id="figure3">
<p><strong>Correlation of replicates of auto-ChIPmentation experiments.</strong> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 10.000 and 5.000 cells was used for the immunoprecipitation with the Diagenode antibody targeting H3K4me3 (Cat. no. C15410003). The experiments were performed on the IP-Star® Compact Automated System.The scatterplots show the correlation between the enrichments between two replicates. The green line represents an ideal case where the two replicates are identical, while the red curve fitted on the data points by a LOESS regression shows the actual trend. A. Correlation analysis between two replicates of auto-ChIPmentation experiments starting from 10.000 cells. B. Correlation analysis between two replicates of auto-ChIPmentation experiments starting from 5.000 cells.</p>
</div>
</li>
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<p>The Auto-ChIPmentation workflow is easy and fast, producing high-quality ChIP-seq data even on low starting amounts.</p>
<center><img src="https://www.diagenode.com/img/product/chipmentation/chipmentation-sequencing-results-for-histone.jpg" width="678" height="240" /></center>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure4"><i class="fa fa-caret-right"></i> Figure 4</a>
<div class="content" id="figure4">
<p><strong>ChIPmentation sequencing results for four histone marks.</strong> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 1,000,000 cells was used for the immunoprecipitation with the Diagenode antibodies targeting H3K4me1 (Cat. no. C15410194), H3K27me3 (Cat. no. C15410195), H3K27ac (Cat. no. C15410196) and IgG (Cat. no. C15410206). The experiment was performed on the IP-Star® Compact Automated System.</p>
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<p><a href="https://www.diagenode.com/en/categories/chip-seq-grade-antibodies">ChIP-seq grade anti-histone antibodies</a> provide high yields with excellent specificity and sensitivity</p>',
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<p>Learn more about: <a href="https://www.diagenode.com/applications/western-blot">Loading control, MW marker visualization</a><em>. <br /></em></p>
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<p><sup><strong>Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong></sup></p>
<p><sup>HeLa cells transfected with a Cas9 expression vector (left) or untransfected cells (right) were fixed in methanol at -20°C, permeabilized with acetone at -20°C and blocked with PBS containing 2% BSA. The cells were stained with the Cas9 C-terminal antibody (Cat. No. C15200229) diluted 1:400, followed by incubation with an anti-mouse secondary antibody coupled to AF488. The bottom images show counter-staining of the nuclei with Hoechst 33342.</sup></p>
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'description' => '<p>Histones are the main protein components of chromatin involved in the compaction of DNA into nucleosomes, the basic units of chromatin. A <strong>nucleosome</strong> consists of one pair of each of the core histones (<strong>H2A</strong>, <strong>H2B</strong>, <strong>H3</strong> and <strong>H4</strong>) forming an octameric structure wrapped by 146 base pairs of DNA. The different nucleosomes are linked by the linker histone<strong> H1, </strong>allowing for further condensation of chromatin.</p>
<p>The core histones have a globular structure with large unstructured N-terminal tails protruding from the nucleosome. They can undergo to multiple post-translational modifications (PTM), mainly at the N-terminal tails. These <strong>post-translational modifications </strong>include methylation, acetylation, phosphorylation, ubiquitinylation, citrullination, sumoylation, deamination and crotonylation. The most well characterized PTMs are <strong>methylation,</strong> <strong>acetylation and phosphorylation</strong>. Histone methylation occurs mainly on lysine (K) residues, which can be mono-, di- or tri-methylated, and on arginines (R), which can be mono-methylated and symmetrically or asymmetrically di-methylated. Histone acetylation occurs on lysines and histone phosphorylation mainly on serines (S), threonines (T) and tyrosines (Y).</p>
<p>The PTMs of the different residues are involved in numerous processes such as DNA repair, DNA replication and chromosome condensation. They influence the chromatin organization and can be positively or negatively associated with gene expression. Trimethylation of H3K4, H3K36 and H3K79, and lysine acetylation generally result in an open chromatin configuration (figure below) and are therefore associated with <strong>euchromatin</strong> and gene activation. Trimethylation of H3K9, K3K27 and H4K20, on the other hand, is enriched in <strong>heterochromatin </strong>and associated with gene silencing. The combination of different histone modifications is called the "<strong>histone code</strong>”, analogous to the genetic code.</p>
<p><img src="https://www.diagenode.com/img/categories/antibodies/histone-marks-illustration.png" /></p>
<p>Diagenode is proud to offer a large range of antibodies against histones and histone modifications. Our antibodies are highly specific and have been validated in many applications, including <strong>ChIP</strong> and <strong>ChIP-seq</strong>.</p>
<p>Diagenode’s collection includes antibodies recognizing:</p>
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<li><strong>Histone H1 variants</strong></li>
<li><strong>Histone H2A, H2A variants and histone H2A</strong> <strong>modifications</strong> (serine phosphorylation, lysine acetylation, lysine ubiquitinylation)</li>
<li><strong>Histone H2B and H2B</strong> <strong>modifications </strong>(serine phosphorylation, lysine acetylation)</li>
<li><strong>Histone H3 and H3 modifications </strong>(lysine methylation (mono-, di- and tri-methylated), lysine acetylation, serine phosphorylation, threonine phosphorylation, arginine methylation (mono-methylated, symmetrically and asymmetrically di-methylated))</li>
<li><strong>Histone H4 and H4 modifications (</strong>lysine methylation (mono-, di- and tri-methylated), lysine acetylation, arginine methylation (mono-methylated and symmetrically di-methylated), serine phosphorylation )</li>
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<p><span style="font-weight: 400;"><strong>HDAC's HAT's, HMT's and other</strong> <strong>enzymes</strong> which modify histones can be found in the category <a href="../categories/chromatin-modifying-proteins-histone-transferase">Histone modifying enzymes</a><br /></span></p>
<p><span style="font-weight: 400;"> Diagenode’s highly validated antibodies:</span></p>
<ul>
<li><span style="font-weight: 400;"> Highly sensitive and specific</span></li>
<li><span style="font-weight: 400;"> Cost-effective (requires less antibody per reaction)</span></li>
<li><span style="font-weight: 400;"> Batch-specific data is available on the website</span></li>
<li><span style="font-weight: 400;"> Expert technical support</span></li>
<li><span style="font-weight: 400;"> Sample sizes available</span></li>
<li><span style="font-weight: 400;"> 100% satisfaction guarantee</span></li>
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<p><span style="font-weight: 400;">Diagenode provides leading solutions for epigenetic research. Because ChIP-seq is a widely-used technique, we validate our antibodies in ChIP and ChIP-seq experiments (in addition to conventional methods like Western blot, Dot blot, ELISA, and immunofluorescence) to provide the highest quality antibody. We standardize our validation and production to guarantee high product quality without technical bias. Diagenode guarantees ChIP-seq grade antibody performance under our suggested conditions.</span></p>
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<p><strong>ChIP-seq profile</strong> of active (H3K4me3 and H3K36me3) and inactive (H3K27me3) marks using Diagenode antibodies.</p>
<img src="https://www.diagenode.com/img/categories/antibodies/chip-seq-grade-antibodies.png" /></div>
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<p><small> ChIP was performed on sheared chromatin from 100,000 K562 cells using iDeal ChIP-seq kit for Histones (cat. No. C01010051) with 1 µg of the Diagenode antibodies against H3K27me3 (cat. No. C15410195) and H3K4me3 (cat. No. C15410003), and 0.5 µg of the antibody against H3K36me3 (cat. No. C15410192). The IP'd DNA was subsequently analysed on an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer's instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. The figure shows the signal distribution along the complete sequence of human chromosome 3, a zoomin to a 10 Mb region and a further zoomin to a 1.5 Mb region. </small></p>
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<p>Diagenode’s highly validated antibodies:</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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<li>100% satisfaction guarantee</li>
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<p><span style="font-weight: 400;">Diagenode’s highly validated antibodies:</span></p>
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<div class="small-12 columns"><center></center>
<p><br />Chromatin immunoprecipitation (<b>ChIP</b>) is a technique to study the associations of proteins with the specific genomic regions in intact cells. One of the most important steps of this protocol is the immunoprecipitation of targeted protein using the antibody specifically recognizing it. The quality of antibodies used in ChIP is essential for the success of the experiment. Diagenode offers extensively validated ChIP-grade antibodies, confirmed for their specificity, and high level of performance in ChIP. Each batch is validated, and batch-specific data are available on the website.</p>
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<p><strong>ChIP results</strong> obtained with the antibody directed against H3K4me3 (Cat. No. <a href="../p/h3k4me3-polyclonal-antibody-premium-50-ug-50-ul">C15410003</a>). </p>
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<div class="small-12 medium-6 large-6 columns"><img src="https://www.diagenode.com/img/product/antibodies/C15410003-fig1-ChIP.jpg" alt="" width="400" height="315" /> </div>
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<p>Our aim at Diagenode is to offer the largest collection of highly specific <strong>ChIP-grade antibodies</strong>. We add new antibodies monthly. Find your ChIP-grade antibody in the list below and check more information about tested applications, extensive validation data, and product information.</p>',
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'description' => '<p style="text-align: justify;"><span>Epigenetic research tools have evolved over time from endpoint PCR to qPCR to the analyses of large sets of genome-wide sequencing data. ChIP sequencing (ChIP-seq) has now become the gold standard method for chromatin studies, given the accuracy and coverage scale of the approach over other methods. Successful ChIP-seq, however, requires a higher level of experimental accuracy and consistency in all steps of ChIP than ever before. Particularly crucial is the quality of ChIP antibodies. </span></p>',
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'description' => '<p>The nuclear PA28γ is known to activate the 20S proteasome, but its precise cellular functions remains unclear. Here, we identify PA28γ as a key factor that structures heterochromatin. We find that in human cells, a fraction of PA28γ-20S proteasome complexes localizes within HP1-linked heterochromatin foci. Our biochemical studies show that PA28γ interacts with HP1 proteins, particularly HP1β, which recruits the PA28γ-20S proteasome complexes to heterochromatin. Loss of PA28γ does not modify the localization of HP1β, its mobility within nuclear foci, or the level of H3K9 tri-methylation, but reduces H4K20 mono- and tri-methylation, modifications involved in heterochromatin establishment. Concordantly, using a quantitative FRET-based microscopy assay to monitor nanometer-scale proximity between nucleosomes in living cells, we find that PA28γ regulates nucleosome proximity within heterochromatin, and thereby controls its compaction. This function of PA28γ is independent of the 20S proteasome. Importantly, HP1β on its own is unable to drive heterochromatin compaction without PA28γ. Combined, our data reveal an unexpected chromatin structural role of PA28γ, and provide new insights into the mechanism that controls HP1β-mediated heterochromatin compaction.</p>',
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'description' => '<p>Following implantation, the naive pluripotent epiblast of the mouse blastocyst generates a rosette, undergoes lumenogenesis and forms the primed pluripotent egg cylinder, which is able to generate the embryonic tissues. How pluripotency progression and morphogenesis are linked and whether intermediate pluripotent states exist remain controversial. We identify here a rosette pluripotent state defined by the co-expression of naive factors with the transcription factor OTX2. Downregulation of blastocyst WNT signals drives the transition into rosette pluripotency by inducing OTX2. The rosette then activates MEK signals that induce lumenogenesis and drive progression to primed pluripotency. Consequently, combined WNT and MEK inhibition supports rosette-like stem cells, a self-renewing naive-primed intermediate. Rosette-like stem cells erase constitutive heterochromatin marks and display a primed chromatin landscape, with bivalently marked primed pluripotency genes. Nonetheless, WNT induces reversion to naive pluripotency. The rosette is therefore a reversible pluripotent intermediate whereby control over both pluripotency progression and morphogenesis pivots from WNT to MEK signals.</p>',
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'description' => '<p>50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input. Single-molecule imaging shows that this step lowers transcription factor residence time and non-specific collisions during sampling for DNA targets. Third, chromatin interactions shift from long range to predominantly short range, and CTCF-mediated loops and contact domains double in numbers. This architectural change facilitates cognate promoter-enhancer contacts and also requires Myc and continual ATP production. Our results thus define the nature and transcriptional impact of chromatin decondensation and reveal an unexpected role for Myc in the establishment of nuclear topology in mammalian cells.</p>',
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<div class="row">
<div class="small-12 columns">
<a href="/jp/p/chipmentation-for-histones-24-rxns"><img src="/img/grey-logo.jpg" alt="default alt" class="th"/></a> </div>
<div class="small-12 columns">
<div class="small-6 columns" style="padding-left:0px;padding-right:0px;margin-top:-6px;margin-left:-1px">
<span class="success label" style="">C01011000</span>
</div>
<div class="small-6 columns text-right" style="padding-left:0px;padding-right:0px;margin-top:-6px">
<!--a href="#" style="color:#B21329"><i class="fa fa-info-circle"></i></a-->
</div>
</div>
<div class="small-12 columns" >
<h6 style="height:60px">Auto ChIPmentation Kit for Histones</h6>
</div>
</div>
</li>
'
$related = array(
'id' => '2914',
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'name' => 'Auto ChIPmentation Kit for Histones - Replaced by C01011009',
'description' => '<div class="extra-spaced"><center></center></div>
<div class="row">
<div class="small-12 medium-8 large-8 columns"><!--<div class="extra-spaced"><center><a href="https://www.diagenode.com/en/pages/grant"><img src="https://www.diagenode.com/img/banners/banner-chipmentation-grant-580x120.jpg" /></a></center></div>-->
<div align="center"><video width="400" height="250" autoplay="autoplay" muted="" loop="loop" controls="controls" src="https://www.diagenode.com/videos/chipmentation-dgo.mp4" frameborder="0" allowfullscreen="allowfullscreen"></video></div>
<div align="center"><a href="https://www.diagenode.com/pages/form-chipmentation" class="center alert radius button"><i class="fa fa-info"></i> Contact us</a></div>
</div>
<div class="small-12 medium-4 large-4 columns"><center><a href="https://www.citeab.com/awards/2018/innovative-product-of-the-year" target="_blank"><img src="https://www.diagenode.com/emailing/images/citeab.png" width="224" height="200" /></a></center></div>
</div>
<p></p>
<p><strong>This product must be used with the <a href="https://www.diagenode.com/en/p/sx-8g-ip-star-compact-automated-system-1-unit">IP-Star Compact Automated System</a>.</strong></p>
<p>Diagenode’s latest technology for histone ChIP-seq, ChIPmentation, incorporates automation with an ligation-free protocol that integrates chromatin immunoprecipitation with NGS library preparation. Using histone ChIPmentation, you can avoid the multi-step ligation required for traditional histone ChIP-seq protocols, providing an easier protocol, faster time to results, and unmatched efficiency and reproducibility for histone ChIP-seq from challenging and low input samples. The ChIPmentaion Kit for Histones is a complete solution, including all buffers for chromatin preparation, immunoprecipitation, tagmentation and multiplexing using 24 single indexes.</p>',
'label1' => 'Characteristics',
'info1' => '<p>ChIPmentation is based on tagmentation that allows library preparation to be integrated during the ChIP itself using transposase and sequencing-compatible adaptors. ChIPmentation involves much easier and shorter protocol with high efficiency for low input samples using optimized ChIP and NGS sample preparation reagents on the IP-Star Compact Automated System. The IP-Star Compact Automated System ensures the highest reproducibility for challenging samples from limited input amounts.</p>
<h3>Benefits of the ChIPmentation system for histone ChIP-seq</h3>
<ul>
<li>Automates ChIP-seq of histones on the IP-Star® for highest reproducibility</li>
<li>Easier and faster than classical ChIP-seq</li>
<li>Validated for various histone marks</li>
<li>Ideal for analysis of large cohorts of samples</li>
<li>Ideal for analysis of large number of marks on a unique sample</li>
<li>High quality sequencing data</li>
</ul>
<p><img src="https://www.diagenode.com/img/product/chipmentation/chipmentation.jpg" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<h3>ChIPmentation - Highest reliability for even the lowest inputs</h3>
<p>The new Auto ChIPmentation technology has been tested on histone marks and compared to available datasets from the ENCODE project (Figure 1). ChIPmentation generated high quality data with low background. In addition, more than 99% of the top 40% peaks obtained with auto-ChIPmentation overlap with ENCODE datasets, which shows that ChIP-seq data obtained with ChIPmentation are highly reliable.</p>
<div class="spaced"><center>A.<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/auto-chipmentation.png" alt="Diagenode-Auto-ChIPmentation" /></center><br /><center>B. Match of the Top40 peaks of Auto-ChIPmentation<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/match-of-the-top40-peaks-of-auto-chipmentation.png" /></center></div>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure1"><i class="fa fa-caret-right"></i> Figure 1</a>
<div class="content" id="figure1">
<p><strong>Comparison of ChIPmentation sequencing results and reference ChIP-seq data.</strong><br /> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 100.000 cells was used for the immunoprecipitation with the Diagenode antibody targeting H3K4me3 (Cat. no. C15410003). The experiment was performed on the IP-Star® Compact Automated System. A. Distribution of the ChIPmentation and ENCODE readsets in a representative region of the genome. B. Comparison of the top 40% peaks from ChIPmentation dataset to the ENCODE dataset.</p>
</div>
</li>
</ul>
<p class="extra-spaced">As ChIPmentation is a ligation-free protocol and requires fewer steps than a classical workflow, it allows the generation of excellent ChIP-seq data from reduced starting amounts. The sequencing data generated from 5,000 and 10,000 cells are very similar to those obtained with 100,000 cells, showing more than 98% overlap of the top 40% peaks (Figure 2).</p>
<center>A.<br /><img class="extra-spaced" style="margin-bottom: 20px;" src="https://www.diagenode.com/img/categories/kits_chromatin_function/chipmentation-sequencing-figure-a.jpg" alt="Diagenode-ChIPmentation-sequencing" /></center>
<div class="row">
<div class="small-6 columns" style="text-align: left;">B. Match of the Top40 peaks of ChIPmentation from 10.000 cells<br /><center><img src="https://www.diagenode.com/img/categories/kits_chromatin_function/match-of-the-top40-peaks-of-chipmentation-from-cent-cells.png" /></center></div>
<div class="small-6 columns">C. Match of the Top40 peaks of ChIPmentation from 5.000 cells<center><img src="https://www.diagenode.com/img/categories/kits_chromatin_function/match-of-the-top40-peaks-of-chipmentation-from-five-cells.png" /></center></div>
</div>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure2"><i class="fa fa-caret-right"></i> Figure 2</a>
<div class="content" id="figure2">
<p><strong>ChIPmentation sequencing results obtained from decreasing starting amounts of cells.</strong><br /> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 100.000, 10.000 and 5.000 cells was used for the immunoprecipitation with the Diagenode antibody targeting H3K4me3 (Cat. no. C15410003). The experiments were performed on the IP-Star® Compact Automated System. A. Distribution of the ChIPmentation readsets in a representative region of the genome. B. and C. Comparison of the top 40% peaks from 10.000 (B.) and 5.000 (C.) cells with dataset generated from 100.000 cells.</p>
</div>
</li>
</ul>
<p class="extra-spaced">Moreover, in order to check the reproducibility of data generated with low cell amounts, correlation between replicates for 10.000 and 5.000 starting cells was performed (Figure 3). The data show very high correlations with Pearson’s coefficients of 0.96 for both conditions.</p>
<div class="row">
<div class="small-6 columns" style="text-align: left;"><center>A. Auto ChIPmentation on 10.000 cells<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/auto-chipmentation-on-10000cells.jpg" caption="false" width="480" height="420" /></center></div>
<div class="small-6 columns"><center>B. Auto ChIPmentation on 5.000 cells<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/auto-chipmentation-on-5000cells.jpg" width="480" height="420" /></center></div>
</div>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure3"><i class="fa fa-caret-right"></i> Figure 3</a>
<div class="content" id="figure3">
<p><strong>Correlation of replicates of auto-ChIPmentation experiments.</strong> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 10.000 and 5.000 cells was used for the immunoprecipitation with the Diagenode antibody targeting H3K4me3 (Cat. no. C15410003). The experiments were performed on the IP-Star® Compact Automated System.The scatterplots show the correlation between the enrichments between two replicates. The green line represents an ideal case where the two replicates are identical, while the red curve fitted on the data points by a LOESS regression shows the actual trend. A. Correlation analysis between two replicates of auto-ChIPmentation experiments starting from 10.000 cells. B. Correlation analysis between two replicates of auto-ChIPmentation experiments starting from 5.000 cells.</p>
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<p>The Auto-ChIPmentation workflow is easy and fast, producing high-quality ChIP-seq data even on low starting amounts.</p>
<center><img src="https://www.diagenode.com/img/product/chipmentation/chipmentation-sequencing-results-for-histone.jpg" width="678" height="240" /></center>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure4"><i class="fa fa-caret-right"></i> Figure 4</a>
<div class="content" id="figure4">
<p><strong>ChIPmentation sequencing results for four histone marks.</strong> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 1,000,000 cells was used for the immunoprecipitation with the Diagenode antibodies targeting H3K4me1 (Cat. no. C15410194), H3K27me3 (Cat. no. C15410195), H3K27ac (Cat. no. C15410196) and IgG (Cat. no. C15410206). The experiment was performed on the IP-Star® Compact Automated System.</p>
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<p><a href="https://www.diagenode.com/en/categories/chip-seq-grade-antibodies">ChIP-seq grade anti-histone antibodies</a> provide high yields with excellent specificity and sensitivity</p>',
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'description' => '<p><span>Polyclonal antibody raised in rabbit against the region of histone H4 containing the trimethylated lysine 20 (H4K20me3), using a KLH-conjugated synthetic peptide. </span></p>',
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIP-a.png" alt="H4K20me3 Antibody ChIP Grade" /></p>
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<p><small><strong>Figure 1. ChIP results obtained with the Diagenode antibody directed against H4K20me3</strong><br />ChIP assays were performed using human HeLa cells, the Diagenode antibody against H4K20me3 (Cat. No. C15410207) and optimized PCR primer pairs for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (Cat. No. C01010051), using sheared chromatin from 1 million cells. The chromatin was spiked with a panel of in vitro assembled nucleosomes, each containing a specific lysine methylation (SNAP-ChIP K-MetStat Panel, Epicypher). A titration consisting of 0.2, 0.5, 1 and 2 µg of antibody per ChIP experiment was analyzed. IgG (1 µg/IP) was used as a negative IP control.<br /> <strong>Figure 1A.</strong> Quantitative PCR was performed with primers specific for the promoter of the active GAPDH and EIF4A2 genes, used as negative controls, and for the ZNF10 gene and the Sat2 satellite repeat, used as positive controls. The graph shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).<br /> <strong>Figure 1B.</strong> Recovery of the nucleosomes carrying the H4K20me1, H4K20me2, H4K2me3 and the unmodified H4K20 as determined by qPCR. The figure clearly shows the antibody is very specific in ChIP for the H4K20me3 modification. </small></p>
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<p>A. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-A.png" alt="H4K20me3 Antibody for ChIP assay" /></p>
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<div class="small-6 columns">
<p><small><strong> Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H4K20me3</strong><br /> ChIP was performed with 0.5 µg of the Diagenode antibody against H4K20me3 (Cat. No. C15410207) on sheared chromatin from 100,000 K562 cells using the “iDeal ChIP-seq” kit. The IP’d DNA was analysed by QPCR as described above (figure 2A). The IP’d DNA was subsequently analysed on an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. Figure 2B shows the signal distribution along the long arm of chromosome 19 and a zoomin to an enriched region containing several ZNF repeat genes. Figure 2C and D show the enrichment in the telomeric region of chromosome 12, also containing several ZNF repeat genes, and at ZNF510 on chromosome 9, respectively. The position of the amplicon used for ChIP-qPCR is indicated by an arrow. </small></p>
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<div class="small-12 columns">
<p class="text-center">B. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-B.png" alt="H4K20me3 Antibody ChIP-seq Grade" /></p>
<p class="text-center">C. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-C.png" alt="H4K20me3 Antibody for ChIP-seq" /></p>
<p class="text-center">D. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-D.png" alt="H4K20me3 Antibody validated in ChIP-seq" /></p>
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<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ELISA.png" alt="H4K20me3 Antibody ELISA validation" /></p>
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<div class="small-6 columns">
<p><small><strong> Figure 3. Determination of the antibody titer</strong><br /> To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H4K20me3 (Cat. No. C15410207) in antigen coated wells. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the antibody was estimated to be 1:21,700. </small></p>
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<div class="row">
<div class="small-4 columns">
<p>A. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_CrossReactivity-A.png" alt="H4K20me3 Antibody Dot Blot validation" /></p>
<p>B. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_CrossReactivity-B.png" alt="H4K20me3 Antibody validated in Peptide Array " /></p>
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<div class="small-8 columns">
<p><small><strong>Figure 4. Cross reactivity tests using the Diagenode antibody directed against H4K20me3</strong><br /> Figure 4A To test the cross reactivity of the Diagenode antibody against H4K20me3 (Cat. No. C15410207), a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H4K20. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 4A shows a high specificity of the antibody for the modification of interest. Figure 4B The specificity of the antibody was further demonstrated by peptide array analyses on an array containing 384 peptides with different combinations of modifications from histone H3, H4, H2A and H2B. The antibody was used at a dilution of 1:10,000. Figure 4B shows the specificity factor, calculated as the ratio of the average intensity of all spots containing the mark, divided by the average intensity of all spots not containing the mark. </small></p>
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<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_WB.png" alt="H4K20me3 Antibody validated in Western Blot" /></p>
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<div class="small-8 columns">
<p><small><strong> Figure 5. Western blot analysis using the Diagenode antibody directed against H4K20me3</strong><br /> Western blot was performed on whole cell (25 µg, lane 1) and histone extracts (15 µg, lane 2) from HeLa cells, and on 1 µg of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the Diagenode antibody against H4K20me3 (Cat. No. C15410207). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left. </small></p>
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<div class="row">
<div class="small-12 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_IF.png" alt="H4K20me3 Antibody validated in Immunofluorescence" /></p>
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<div class="row">
<div class="small-12 columns">
<p><small><strong> Figure 6. Immunofluorescence using the Diagenode antibody directed against H4K20me3</strong><br /> HeLa cells were stained with the Diagenode antibody against H4K20me3 (Cat. No. C15410207) and with DAPI. Cells were fixed with methanol and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H4K20me3 antibody (left) diluted 1:500 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right. </small></p>
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'description' => '<p><span>Polyclonal antibody raised in rabbit against the region of histone H4 containing the trimethylated lysine 20 (H4K20me3), using a KLH-conjugated synthetic peptide. </span></p>',
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<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIP-a.png" alt="H4K20me3 Antibody ChIP Grade" /></p>
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<p><small><strong>Figure 1. ChIP results obtained with the Diagenode antibody directed against H4K20me3</strong><br />ChIP assays were performed using human HeLa cells, the Diagenode antibody against H4K20me3 (Cat. No. C15410207) and optimized PCR primer pairs for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (Cat. No. C01010051), using sheared chromatin from 1 million cells. The chromatin was spiked with a panel of in vitro assembled nucleosomes, each containing a specific lysine methylation (SNAP-ChIP K-MetStat Panel, Epicypher). A titration consisting of 0.2, 0.5, 1 and 2 µg of antibody per ChIP experiment was analyzed. IgG (1 µg/IP) was used as a negative IP control.<br /> <strong>Figure 1A.</strong> Quantitative PCR was performed with primers specific for the promoter of the active GAPDH and EIF4A2 genes, used as negative controls, and for the ZNF10 gene and the Sat2 satellite repeat, used as positive controls. The graph shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).<br /> <strong>Figure 1B.</strong> Recovery of the nucleosomes carrying the H4K20me1, H4K20me2, H4K2me3 and the unmodified H4K20 as determined by qPCR. The figure clearly shows the antibody is very specific in ChIP for the H4K20me3 modification. </small></p>
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<p>A. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-A.png" alt="H4K20me3 Antibody for ChIP assay" /></p>
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<div class="small-6 columns">
<p><small><strong> Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H4K20me3</strong><br /> ChIP was performed with 0.5 µg of the Diagenode antibody against H4K20me3 (Cat. No. C15410207) on sheared chromatin from 100,000 K562 cells using the “iDeal ChIP-seq” kit. The IP’d DNA was analysed by QPCR as described above (figure 2A). The IP’d DNA was subsequently analysed on an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. Figure 2B shows the signal distribution along the long arm of chromosome 19 and a zoomin to an enriched region containing several ZNF repeat genes. Figure 2C and D show the enrichment in the telomeric region of chromosome 12, also containing several ZNF repeat genes, and at ZNF510 on chromosome 9, respectively. The position of the amplicon used for ChIP-qPCR is indicated by an arrow. </small></p>
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<div class="row">
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<p class="text-center">B. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-B.png" alt="H4K20me3 Antibody ChIP-seq Grade" /></p>
<p class="text-center">C. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-C.png" alt="H4K20me3 Antibody for ChIP-seq" /></p>
<p class="text-center">D. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-D.png" alt="H4K20me3 Antibody validated in ChIP-seq" /></p>
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<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ELISA.png" alt="H4K20me3 Antibody ELISA validation" /></p>
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<div class="small-6 columns">
<p><small><strong> Figure 3. Determination of the antibody titer</strong><br /> To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H4K20me3 (Cat. No. C15410207) in antigen coated wells. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the antibody was estimated to be 1:21,700. </small></p>
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<div class="row">
<div class="small-4 columns">
<p>A. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_CrossReactivity-A.png" alt="H4K20me3 Antibody Dot Blot validation" /></p>
<p>B. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_CrossReactivity-B.png" alt="H4K20me3 Antibody validated in Peptide Array " /></p>
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<div class="small-8 columns">
<p><small><strong>Figure 4. Cross reactivity tests using the Diagenode antibody directed against H4K20me3</strong><br /> Figure 4A To test the cross reactivity of the Diagenode antibody against H4K20me3 (Cat. No. C15410207), a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H4K20. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 4A shows a high specificity of the antibody for the modification of interest. Figure 4B The specificity of the antibody was further demonstrated by peptide array analyses on an array containing 384 peptides with different combinations of modifications from histone H3, H4, H2A and H2B. The antibody was used at a dilution of 1:10,000. Figure 4B shows the specificity factor, calculated as the ratio of the average intensity of all spots containing the mark, divided by the average intensity of all spots not containing the mark. </small></p>
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<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_WB.png" alt="H4K20me3 Antibody validated in Western Blot" /></p>
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<div class="small-8 columns">
<p><small><strong> Figure 5. Western blot analysis using the Diagenode antibody directed against H4K20me3</strong><br /> Western blot was performed on whole cell (25 µg, lane 1) and histone extracts (15 µg, lane 2) from HeLa cells, and on 1 µg of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the Diagenode antibody against H4K20me3 (Cat. No. C15410207). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left. </small></p>
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</div>
<div class="row">
<div class="small-12 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_IF.png" alt="H4K20me3 Antibody validated in Immunofluorescence" /></p>
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<div class="row">
<div class="small-12 columns">
<p><small><strong> Figure 6. Immunofluorescence using the Diagenode antibody directed against H4K20me3</strong><br /> HeLa cells were stained with the Diagenode antibody against H4K20me3 (Cat. No. C15410207) and with DAPI. Cells were fixed with methanol and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H4K20me3 antibody (left) diluted 1:500 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right. </small></p>
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'description' => 'Histones are the main constituents of the protein part of chromosomes of eukaryotic cells. They are rich in the amino acids arginine and lysine and have been greatly conserved during evolution. Histones pack the DNA into tight masses of chromatin. Two core histones of each class H2A, H2B, H3 and H4 assemble and are wrapped by 146 base pairs of DNA to form one octameric nucleosome. Histone tails undergo numerous post-translational modifications, which either directly or indirectly alter chromatin structure to facilitate transcriptional activation or repression or other nuclear processes. In addition to the genetic code, combinations of the different histone modifications reveal the so-called “histone code”. Histone methylation and demethylation is dynamically regulated by respectively histone methyl transferases and histone demethylases. Trimethylation of histone H4K20 is associated with inactive genomic regions, satellite repeats and ZNF gene repeats.',
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<thead>
<tr>
<th>Applications</th>
<th>Suggested dilution</th>
<th>References</th>
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<tbody>
<tr>
<td>ChIP/ChIP-seq <sup>*</sup></td>
<td>0.5-1 µg per IP</td>
<td>Fig 1, 2</td>
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<tr>
<td>ELISA</td>
<td>1:500</td>
<td>Fig 3</td>
</tr>
<tr>
<td>Dot Blotting/Peptide array</td>
<td>1:20,000/1:10,000</td>
<td>Fig 4</td>
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<tr>
<td>Western Blotting</td>
<td>1:1,000</td>
<td>Fig 5</td>
</tr>
<tr>
<td>Immunofluorescence</td>
<td>1:500</td>
<td>Fig 6</td>
</tr>
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<p></p>
<p><small><sup>*</sup> Please note that the optimal antibody amount per IP should be determined by the end-user. We recommend testing 0.5-5 µg per IP.</small></p>',
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'description' => '<p><span>Polyclonal antibody raised in rabbit against the region of histone <strong>H4 containing the trimethylated lysine 20 (H4K20me3)</strong>, using a KLH-conjugated synthetic peptide. </span></p>',
'label1' => 'Validation data',
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<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIP-a.png" alt="H4K20me3 Antibody ChIP Grade" /></p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIP-b.png" alt="H4K20me3 Antibody for ChIP" /></p>
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<div class="small-6 columns">
<p><small><strong>Figure 1. ChIP results obtained with the Diagenode antibody directed against H4K20me3</strong><br />ChIP assays were performed using human HeLa cells, the Diagenode antibody against H4K20me3 (Cat. No. C15410207) and optimized PCR primer pairs for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (Cat. No. C01010051), using sheared chromatin from 1 million cells. The chromatin was spiked with a panel of in vitro assembled nucleosomes, each containing a specific lysine methylation (SNAP-ChIP K-MetStat Panel, Epicypher). A titration consisting of 0.2, 0.5, 1 and 2 µg of antibody per ChIP experiment was analyzed. IgG (1 µg/IP) was used as a negative IP control.<br /> <strong>Figure 1A.</strong> Quantitative PCR was performed with primers specific for the promoter of the active GAPDH and EIF4A2 genes, used as negative controls, and for the ZNF10 gene and the Sat2 satellite repeat, used as positive controls. The graph shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).<br /> <strong>Figure 1B.</strong> Recovery of the nucleosomes carrying the H4K20me1, H4K20me2, H4K2me3 and the unmodified H4K20 as determined by qPCR. The figure clearly shows the antibody is very specific in ChIP for the H4K20me3 modification. </small></p>
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<div class="extra-spaced"></div>
<div class="extra-spaced"></div>
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<p>A. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-A.png" alt="H4K20me3 Antibody for ChIP assay" /></p>
</div>
<div class="small-6 columns">
<p><small><strong> Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H4K20me3</strong><br /> ChIP was performed with 0.5 µg of the Diagenode antibody against H4K20me3 (Cat. No. C15410207) on sheared chromatin from 100,000 K562 cells using the “iDeal ChIP-seq” kit. The IP’d DNA was analysed by QPCR as described above (figure 2A). The IP’d DNA was subsequently analysed on an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. Figure 2B shows the signal distribution along the long arm of chromosome 19 and a zoomin to an enriched region containing several ZNF repeat genes. Figure 2C and D show the enrichment in the telomeric region of chromosome 12, also containing several ZNF repeat genes, and at ZNF510 on chromosome 9, respectively. The position of the amplicon used for ChIP-qPCR is indicated by an arrow. </small></p>
</div>
</div>
<div class="row">
<div class="small-12 columns">
<p class="text-center">B. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-B.png" alt="H4K20me3 Antibody ChIP-seq Grade" /></p>
<p class="text-center">C. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-C.png" alt="H4K20me3 Antibody for ChIP-seq" /></p>
<p class="text-center">D. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ChIPseq-D.png" alt="H4K20me3 Antibody validated in ChIP-seq" /></p>
</div>
</div>
<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_ELISA.png" alt="H4K20me3 Antibody ELISA validation" /></p>
</div>
<div class="small-6 columns">
<p><small><strong> Figure 3. Determination of the antibody titer</strong><br /> To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H4K20me3 (Cat. No. C15410207) in antigen coated wells. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the antibody was estimated to be 1:21,700. </small></p>
</div>
</div>
<div class="row">
<div class="small-4 columns">
<p>A. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_CrossReactivity-A.png" alt="H4K20me3 Antibody Dot Blot validation" /></p>
<p>B. <img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_CrossReactivity-B.png" alt="H4K20me3 Antibody validated in Peptide Array " /></p>
</div>
<div class="small-8 columns">
<p><small><strong>Figure 4. Cross reactivity tests using the Diagenode antibody directed against H4K20me3</strong><br /> Figure 4A To test the cross reactivity of the Diagenode antibody against H4K20me3 (Cat. No. C15410207), a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H4K20. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 4A shows a high specificity of the antibody for the modification of interest. Figure 4B The specificity of the antibody was further demonstrated by peptide array analyses on an array containing 384 peptides with different combinations of modifications from histone H3, H4, H2A and H2B. The antibody was used at a dilution of 1:10,000. Figure 4B shows the specificity factor, calculated as the ratio of the average intensity of all spots containing the mark, divided by the average intensity of all spots not containing the mark. </small></p>
</div>
</div>
<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_WB.png" alt="H4K20me3 Antibody validated in Western Blot" /></p>
</div>
<div class="small-8 columns">
<p><small><strong> Figure 5. Western blot analysis using the Diagenode antibody directed against H4K20me3</strong><br /> Western blot was performed on whole cell (25 µg, lane 1) and histone extracts (15 µg, lane 2) from HeLa cells, and on 1 µg of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the Diagenode antibody against H4K20me3 (Cat. No. C15410207). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left. </small></p>
</div>
</div>
<div class="row">
<div class="small-12 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15410207_A2730P_IF.png" alt="H4K20me3 Antibody validated in Immunofluorescence" /></p>
</div>
</div>
<div class="row">
<div class="small-12 columns">
<p><small><strong> Figure 6. Immunofluorescence using the Diagenode antibody directed against H4K20me3</strong><br /> HeLa cells were stained with the Diagenode antibody against H4K20me3 (Cat. No. C15410207) and with DAPI. Cells were fixed with methanol and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H4K20me3 antibody (left) diluted 1:500 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right. </small></p>
</div>
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'meta_title' => 'H4K20me3 Antibody - ChIP-seq Grade (C15410207) | Diagenode',
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'meta_description' => 'H4K20me3 (Histone H4 trimethylated at lysine 20) Polyclonal Antibody validated in ChIP-seq, ChIP-qPCR, ELISA, DB, WB and IF. Specificity confirmed by Peptide array assay. Batch-specific data available on the website. Sample size available.',
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'name' => 'Auto ChIPmentation Kit for Histones - Replaced by C01011009',
'description' => '<div class="extra-spaced"><center></center></div>
<div class="row">
<div class="small-12 medium-8 large-8 columns"><!--<div class="extra-spaced"><center><a href="https://www.diagenode.com/en/pages/grant"><img src="https://www.diagenode.com/img/banners/banner-chipmentation-grant-580x120.jpg" /></a></center></div>-->
<div align="center"><video width="400" height="250" autoplay="autoplay" muted="" loop="loop" controls="controls" src="https://www.diagenode.com/videos/chipmentation-dgo.mp4" frameborder="0" allowfullscreen="allowfullscreen"></video></div>
<div align="center"><a href="https://www.diagenode.com/pages/form-chipmentation" class="center alert radius button"><i class="fa fa-info"></i> Contact us</a></div>
</div>
<div class="small-12 medium-4 large-4 columns"><center><a href="https://www.citeab.com/awards/2018/innovative-product-of-the-year" target="_blank"><img src="https://www.diagenode.com/emailing/images/citeab.png" width="224" height="200" /></a></center></div>
</div>
<p></p>
<p><strong>This product must be used with the <a href="https://www.diagenode.com/en/p/sx-8g-ip-star-compact-automated-system-1-unit">IP-Star Compact Automated System</a>.</strong></p>
<p>Diagenode’s latest technology for histone ChIP-seq, ChIPmentation, incorporates automation with an ligation-free protocol that integrates chromatin immunoprecipitation with NGS library preparation. Using histone ChIPmentation, you can avoid the multi-step ligation required for traditional histone ChIP-seq protocols, providing an easier protocol, faster time to results, and unmatched efficiency and reproducibility for histone ChIP-seq from challenging and low input samples. The ChIPmentaion Kit for Histones is a complete solution, including all buffers for chromatin preparation, immunoprecipitation, tagmentation and multiplexing using 24 single indexes.</p>',
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'info1' => '<p>ChIPmentation is based on tagmentation that allows library preparation to be integrated during the ChIP itself using transposase and sequencing-compatible adaptors. ChIPmentation involves much easier and shorter protocol with high efficiency for low input samples using optimized ChIP and NGS sample preparation reagents on the IP-Star Compact Automated System. The IP-Star Compact Automated System ensures the highest reproducibility for challenging samples from limited input amounts.</p>
<h3>Benefits of the ChIPmentation system for histone ChIP-seq</h3>
<ul>
<li>Automates ChIP-seq of histones on the IP-Star® for highest reproducibility</li>
<li>Easier and faster than classical ChIP-seq</li>
<li>Validated for various histone marks</li>
<li>Ideal for analysis of large cohorts of samples</li>
<li>Ideal for analysis of large number of marks on a unique sample</li>
<li>High quality sequencing data</li>
</ul>
<p><img src="https://www.diagenode.com/img/product/chipmentation/chipmentation.jpg" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<h3>ChIPmentation - Highest reliability for even the lowest inputs</h3>
<p>The new Auto ChIPmentation technology has been tested on histone marks and compared to available datasets from the ENCODE project (Figure 1). ChIPmentation generated high quality data with low background. In addition, more than 99% of the top 40% peaks obtained with auto-ChIPmentation overlap with ENCODE datasets, which shows that ChIP-seq data obtained with ChIPmentation are highly reliable.</p>
<div class="spaced"><center>A.<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/auto-chipmentation.png" alt="Diagenode-Auto-ChIPmentation" /></center><br /><center>B. Match of the Top40 peaks of Auto-ChIPmentation<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/match-of-the-top40-peaks-of-auto-chipmentation.png" /></center></div>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure1"><i class="fa fa-caret-right"></i> Figure 1</a>
<div class="content" id="figure1">
<p><strong>Comparison of ChIPmentation sequencing results and reference ChIP-seq data.</strong><br /> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 100.000 cells was used for the immunoprecipitation with the Diagenode antibody targeting H3K4me3 (Cat. no. C15410003). The experiment was performed on the IP-Star® Compact Automated System. A. Distribution of the ChIPmentation and ENCODE readsets in a representative region of the genome. B. Comparison of the top 40% peaks from ChIPmentation dataset to the ENCODE dataset.</p>
</div>
</li>
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<p class="extra-spaced">As ChIPmentation is a ligation-free protocol and requires fewer steps than a classical workflow, it allows the generation of excellent ChIP-seq data from reduced starting amounts. The sequencing data generated from 5,000 and 10,000 cells are very similar to those obtained with 100,000 cells, showing more than 98% overlap of the top 40% peaks (Figure 2).</p>
<center>A.<br /><img class="extra-spaced" style="margin-bottom: 20px;" src="https://www.diagenode.com/img/categories/kits_chromatin_function/chipmentation-sequencing-figure-a.jpg" alt="Diagenode-ChIPmentation-sequencing" /></center>
<div class="row">
<div class="small-6 columns" style="text-align: left;">B. Match of the Top40 peaks of ChIPmentation from 10.000 cells<br /><center><img src="https://www.diagenode.com/img/categories/kits_chromatin_function/match-of-the-top40-peaks-of-chipmentation-from-cent-cells.png" /></center></div>
<div class="small-6 columns">C. Match of the Top40 peaks of ChIPmentation from 5.000 cells<center><img src="https://www.diagenode.com/img/categories/kits_chromatin_function/match-of-the-top40-peaks-of-chipmentation-from-five-cells.png" /></center></div>
</div>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure2"><i class="fa fa-caret-right"></i> Figure 2</a>
<div class="content" id="figure2">
<p><strong>ChIPmentation sequencing results obtained from decreasing starting amounts of cells.</strong><br /> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 100.000, 10.000 and 5.000 cells was used for the immunoprecipitation with the Diagenode antibody targeting H3K4me3 (Cat. no. C15410003). The experiments were performed on the IP-Star® Compact Automated System. A. Distribution of the ChIPmentation readsets in a representative region of the genome. B. and C. Comparison of the top 40% peaks from 10.000 (B.) and 5.000 (C.) cells with dataset generated from 100.000 cells.</p>
</div>
</li>
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<p class="extra-spaced">Moreover, in order to check the reproducibility of data generated with low cell amounts, correlation between replicates for 10.000 and 5.000 starting cells was performed (Figure 3). The data show very high correlations with Pearson’s coefficients of 0.96 for both conditions.</p>
<div class="row">
<div class="small-6 columns" style="text-align: left;"><center>A. Auto ChIPmentation on 10.000 cells<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/auto-chipmentation-on-10000cells.jpg" caption="false" width="480" height="420" /></center></div>
<div class="small-6 columns"><center>B. Auto ChIPmentation on 5.000 cells<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/auto-chipmentation-on-5000cells.jpg" width="480" height="420" /></center></div>
</div>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure3"><i class="fa fa-caret-right"></i> Figure 3</a>
<div class="content" id="figure3">
<p><strong>Correlation of replicates of auto-ChIPmentation experiments.</strong> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 10.000 and 5.000 cells was used for the immunoprecipitation with the Diagenode antibody targeting H3K4me3 (Cat. no. C15410003). The experiments were performed on the IP-Star® Compact Automated System.The scatterplots show the correlation between the enrichments between two replicates. The green line represents an ideal case where the two replicates are identical, while the red curve fitted on the data points by a LOESS regression shows the actual trend. A. Correlation analysis between two replicates of auto-ChIPmentation experiments starting from 10.000 cells. B. Correlation analysis between two replicates of auto-ChIPmentation experiments starting from 5.000 cells.</p>
</div>
</li>
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<p>The Auto-ChIPmentation workflow is easy and fast, producing high-quality ChIP-seq data even on low starting amounts.</p>
<center><img src="https://www.diagenode.com/img/product/chipmentation/chipmentation-sequencing-results-for-histone.jpg" width="678" height="240" /></center>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure4"><i class="fa fa-caret-right"></i> Figure 4</a>
<div class="content" id="figure4">
<p><strong>ChIPmentation sequencing results for four histone marks.</strong> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 1,000,000 cells was used for the immunoprecipitation with the Diagenode antibodies targeting H3K4me1 (Cat. no. C15410194), H3K27me3 (Cat. no. C15410195), H3K27ac (Cat. no. C15410196) and IgG (Cat. no. C15410206). The experiment was performed on the IP-Star® Compact Automated System.</p>
</div>
</li>
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<p><a href="https://www.diagenode.com/en/categories/chip-seq-grade-antibodies">ChIP-seq grade anti-histone antibodies</a> provide high yields with excellent specificity and sensitivity</p>',
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<p>Learn more about: <a href="https://www.diagenode.com/applications/western-blot">Loading control, MW marker visualization</a><em>. <br /></em></p>
<p><em></em>Check our selection of antibodies validated in Western blot.</p>',
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<p>Diagenode offers huge selection of highly sensitive antibodies validated in IF.</p>
<p><img src="https://www.diagenode.com/img/product/antibodies/C15200229-IF.jpg" alt="" height="245" width="256" /></p>
<p><sup><strong>Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9</strong></sup></p>
<p><sup>HeLa cells transfected with a Cas9 expression vector (left) or untransfected cells (right) were fixed in methanol at -20°C, permeabilized with acetone at -20°C and blocked with PBS containing 2% BSA. The cells were stained with the Cas9 C-terminal antibody (Cat. No. C15200229) diluted 1:400, followed by incubation with an anti-mouse secondary antibody coupled to AF488. The bottom images show counter-staining of the nuclei with Hoechst 33342.</sup></p>
<h5><sup>Check our selection of antibodies validated in IF.</sup></h5>',
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'description' => '<p>Histones are the main protein components of chromatin involved in the compaction of DNA into nucleosomes, the basic units of chromatin. A <strong>nucleosome</strong> consists of one pair of each of the core histones (<strong>H2A</strong>, <strong>H2B</strong>, <strong>H3</strong> and <strong>H4</strong>) forming an octameric structure wrapped by 146 base pairs of DNA. The different nucleosomes are linked by the linker histone<strong> H1, </strong>allowing for further condensation of chromatin.</p>
<p>The core histones have a globular structure with large unstructured N-terminal tails protruding from the nucleosome. They can undergo to multiple post-translational modifications (PTM), mainly at the N-terminal tails. These <strong>post-translational modifications </strong>include methylation, acetylation, phosphorylation, ubiquitinylation, citrullination, sumoylation, deamination and crotonylation. The most well characterized PTMs are <strong>methylation,</strong> <strong>acetylation and phosphorylation</strong>. Histone methylation occurs mainly on lysine (K) residues, which can be mono-, di- or tri-methylated, and on arginines (R), which can be mono-methylated and symmetrically or asymmetrically di-methylated. Histone acetylation occurs on lysines and histone phosphorylation mainly on serines (S), threonines (T) and tyrosines (Y).</p>
<p>The PTMs of the different residues are involved in numerous processes such as DNA repair, DNA replication and chromosome condensation. They influence the chromatin organization and can be positively or negatively associated with gene expression. Trimethylation of H3K4, H3K36 and H3K79, and lysine acetylation generally result in an open chromatin configuration (figure below) and are therefore associated with <strong>euchromatin</strong> and gene activation. Trimethylation of H3K9, K3K27 and H4K20, on the other hand, is enriched in <strong>heterochromatin </strong>and associated with gene silencing. The combination of different histone modifications is called the "<strong>histone code</strong>”, analogous to the genetic code.</p>
<p><img src="https://www.diagenode.com/img/categories/antibodies/histone-marks-illustration.png" /></p>
<p>Diagenode is proud to offer a large range of antibodies against histones and histone modifications. Our antibodies are highly specific and have been validated in many applications, including <strong>ChIP</strong> and <strong>ChIP-seq</strong>.</p>
<p>Diagenode’s collection includes antibodies recognizing:</p>
<ul>
<li><strong>Histone H1 variants</strong></li>
<li><strong>Histone H2A, H2A variants and histone H2A</strong> <strong>modifications</strong> (serine phosphorylation, lysine acetylation, lysine ubiquitinylation)</li>
<li><strong>Histone H2B and H2B</strong> <strong>modifications </strong>(serine phosphorylation, lysine acetylation)</li>
<li><strong>Histone H3 and H3 modifications </strong>(lysine methylation (mono-, di- and tri-methylated), lysine acetylation, serine phosphorylation, threonine phosphorylation, arginine methylation (mono-methylated, symmetrically and asymmetrically di-methylated))</li>
<li><strong>Histone H4 and H4 modifications (</strong>lysine methylation (mono-, di- and tri-methylated), lysine acetylation, arginine methylation (mono-methylated and symmetrically di-methylated), serine phosphorylation )</li>
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<p><span style="font-weight: 400;"><strong>HDAC's HAT's, HMT's and other</strong> <strong>enzymes</strong> which modify histones can be found in the category <a href="../categories/chromatin-modifying-proteins-histone-transferase">Histone modifying enzymes</a><br /></span></p>
<p><span style="font-weight: 400;"> Diagenode’s highly validated antibodies:</span></p>
<ul>
<li><span style="font-weight: 400;"> Highly sensitive and specific</span></li>
<li><span style="font-weight: 400;"> Cost-effective (requires less antibody per reaction)</span></li>
<li><span style="font-weight: 400;"> Batch-specific data is available on the website</span></li>
<li><span style="font-weight: 400;"> Expert technical support</span></li>
<li><span style="font-weight: 400;"> Sample sizes available</span></li>
<li><span style="font-weight: 400;"> 100% satisfaction guarantee</span></li>
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<p><span style="font-weight: 400;">Diagenode provides leading solutions for epigenetic research. Because ChIP-seq is a widely-used technique, we validate our antibodies in ChIP and ChIP-seq experiments (in addition to conventional methods like Western blot, Dot blot, ELISA, and immunofluorescence) to provide the highest quality antibody. We standardize our validation and production to guarantee high product quality without technical bias. Diagenode guarantees ChIP-seq grade antibody performance under our suggested conditions.</span></p>
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<p><strong>ChIP-seq profile</strong> of active (H3K4me3 and H3K36me3) and inactive (H3K27me3) marks using Diagenode antibodies.</p>
<img src="https://www.diagenode.com/img/categories/antibodies/chip-seq-grade-antibodies.png" /></div>
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<p><small> ChIP was performed on sheared chromatin from 100,000 K562 cells using iDeal ChIP-seq kit for Histones (cat. No. C01010051) with 1 µg of the Diagenode antibodies against H3K27me3 (cat. No. C15410195) and H3K4me3 (cat. No. C15410003), and 0.5 µg of the antibody against H3K36me3 (cat. No. C15410192). The IP'd DNA was subsequently analysed on an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer's instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. The figure shows the signal distribution along the complete sequence of human chromosome 3, a zoomin to a 10 Mb region and a further zoomin to a 1.5 Mb region. </small></p>
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<p>Diagenode’s highly validated antibodies:</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>
<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>
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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>
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<p><br />Chromatin immunoprecipitation (<b>ChIP</b>) is a technique to study the associations of proteins with the specific genomic regions in intact cells. One of the most important steps of this protocol is the immunoprecipitation of targeted protein using the antibody specifically recognizing it. The quality of antibodies used in ChIP is essential for the success of the experiment. Diagenode offers extensively validated ChIP-grade antibodies, confirmed for their specificity, and high level of performance in ChIP. Each batch is validated, and batch-specific data are available on the website.</p>
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<p><strong>ChIP results</strong> obtained with the antibody directed against H3K4me3 (Cat. No. <a href="../p/h3k4me3-polyclonal-antibody-premium-50-ug-50-ul">C15410003</a>). </p>
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<p>Our aim at Diagenode is to offer the largest collection of highly specific <strong>ChIP-grade antibodies</strong>. We add new antibodies monthly. Find your ChIP-grade antibody in the list below and check more information about tested applications, extensive validation data, and product information.</p>',
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$related_products = '<li>
<div class="row">
<div class="small-12 columns">
<a href="/jp/p/chipmentation-for-histones-24-rxns"><img src="/img/grey-logo.jpg" alt="default alt" class="th"/></a> </div>
<div class="small-12 columns">
<div class="small-6 columns" style="padding-left:0px;padding-right:0px;margin-top:-6px;margin-left:-1px">
<span class="success label" style="">C01011000</span>
</div>
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<!--a href="#" style="color:#B21329"><i class="fa fa-info-circle"></i></a-->
</div>
</div>
<div class="small-12 columns" >
<h6 style="height:60px">Auto ChIPmentation Kit for Histones</h6>
</div>
</div>
</li>
'
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'id' => '2914',
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'name' => 'Auto ChIPmentation Kit for Histones - Replaced by C01011009',
'description' => '<div class="extra-spaced"><center></center></div>
<div class="row">
<div class="small-12 medium-8 large-8 columns"><!--<div class="extra-spaced"><center><a href="https://www.diagenode.com/en/pages/grant"><img src="https://www.diagenode.com/img/banners/banner-chipmentation-grant-580x120.jpg" /></a></center></div>-->
<div align="center"><video width="400" height="250" autoplay="autoplay" muted="" loop="loop" controls="controls" src="https://www.diagenode.com/videos/chipmentation-dgo.mp4" frameborder="0" allowfullscreen="allowfullscreen"></video></div>
<div align="center"><a href="https://www.diagenode.com/pages/form-chipmentation" class="center alert radius button"><i class="fa fa-info"></i> Contact us</a></div>
</div>
<div class="small-12 medium-4 large-4 columns"><center><a href="https://www.citeab.com/awards/2018/innovative-product-of-the-year" target="_blank"><img src="https://www.diagenode.com/emailing/images/citeab.png" width="224" height="200" /></a></center></div>
</div>
<p></p>
<p><strong>This product must be used with the <a href="https://www.diagenode.com/en/p/sx-8g-ip-star-compact-automated-system-1-unit">IP-Star Compact Automated System</a>.</strong></p>
<p>Diagenode’s latest technology for histone ChIP-seq, ChIPmentation, incorporates automation with an ligation-free protocol that integrates chromatin immunoprecipitation with NGS library preparation. Using histone ChIPmentation, you can avoid the multi-step ligation required for traditional histone ChIP-seq protocols, providing an easier protocol, faster time to results, and unmatched efficiency and reproducibility for histone ChIP-seq from challenging and low input samples. The ChIPmentaion Kit for Histones is a complete solution, including all buffers for chromatin preparation, immunoprecipitation, tagmentation and multiplexing using 24 single indexes.</p>',
'label1' => 'Characteristics',
'info1' => '<p>ChIPmentation is based on tagmentation that allows library preparation to be integrated during the ChIP itself using transposase and sequencing-compatible adaptors. ChIPmentation involves much easier and shorter protocol with high efficiency for low input samples using optimized ChIP and NGS sample preparation reagents on the IP-Star Compact Automated System. The IP-Star Compact Automated System ensures the highest reproducibility for challenging samples from limited input amounts.</p>
<h3>Benefits of the ChIPmentation system for histone ChIP-seq</h3>
<ul>
<li>Automates ChIP-seq of histones on the IP-Star® for highest reproducibility</li>
<li>Easier and faster than classical ChIP-seq</li>
<li>Validated for various histone marks</li>
<li>Ideal for analysis of large cohorts of samples</li>
<li>Ideal for analysis of large number of marks on a unique sample</li>
<li>High quality sequencing data</li>
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<p><img src="https://www.diagenode.com/img/product/chipmentation/chipmentation.jpg" style="display: block; margin-left: auto; margin-right: auto;" /></p>
<h3>ChIPmentation - Highest reliability for even the lowest inputs</h3>
<p>The new Auto ChIPmentation technology has been tested on histone marks and compared to available datasets from the ENCODE project (Figure 1). ChIPmentation generated high quality data with low background. In addition, more than 99% of the top 40% peaks obtained with auto-ChIPmentation overlap with ENCODE datasets, which shows that ChIP-seq data obtained with ChIPmentation are highly reliable.</p>
<div class="spaced"><center>A.<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/auto-chipmentation.png" alt="Diagenode-Auto-ChIPmentation" /></center><br /><center>B. Match of the Top40 peaks of Auto-ChIPmentation<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/match-of-the-top40-peaks-of-auto-chipmentation.png" /></center></div>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure1"><i class="fa fa-caret-right"></i> Figure 1</a>
<div class="content" id="figure1">
<p><strong>Comparison of ChIPmentation sequencing results and reference ChIP-seq data.</strong><br /> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 100.000 cells was used for the immunoprecipitation with the Diagenode antibody targeting H3K4me3 (Cat. no. C15410003). The experiment was performed on the IP-Star® Compact Automated System. A. Distribution of the ChIPmentation and ENCODE readsets in a representative region of the genome. B. Comparison of the top 40% peaks from ChIPmentation dataset to the ENCODE dataset.</p>
</div>
</li>
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<p class="extra-spaced">As ChIPmentation is a ligation-free protocol and requires fewer steps than a classical workflow, it allows the generation of excellent ChIP-seq data from reduced starting amounts. The sequencing data generated from 5,000 and 10,000 cells are very similar to those obtained with 100,000 cells, showing more than 98% overlap of the top 40% peaks (Figure 2).</p>
<center>A.<br /><img class="extra-spaced" style="margin-bottom: 20px;" src="https://www.diagenode.com/img/categories/kits_chromatin_function/chipmentation-sequencing-figure-a.jpg" alt="Diagenode-ChIPmentation-sequencing" /></center>
<div class="row">
<div class="small-6 columns" style="text-align: left;">B. Match of the Top40 peaks of ChIPmentation from 10.000 cells<br /><center><img src="https://www.diagenode.com/img/categories/kits_chromatin_function/match-of-the-top40-peaks-of-chipmentation-from-cent-cells.png" /></center></div>
<div class="small-6 columns">C. Match of the Top40 peaks of ChIPmentation from 5.000 cells<center><img src="https://www.diagenode.com/img/categories/kits_chromatin_function/match-of-the-top40-peaks-of-chipmentation-from-five-cells.png" /></center></div>
</div>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure2"><i class="fa fa-caret-right"></i> Figure 2</a>
<div class="content" id="figure2">
<p><strong>ChIPmentation sequencing results obtained from decreasing starting amounts of cells.</strong><br /> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 100.000, 10.000 and 5.000 cells was used for the immunoprecipitation with the Diagenode antibody targeting H3K4me3 (Cat. no. C15410003). The experiments were performed on the IP-Star® Compact Automated System. A. Distribution of the ChIPmentation readsets in a representative region of the genome. B. and C. Comparison of the top 40% peaks from 10.000 (B.) and 5.000 (C.) cells with dataset generated from 100.000 cells.</p>
</div>
</li>
</ul>
<p class="extra-spaced">Moreover, in order to check the reproducibility of data generated with low cell amounts, correlation between replicates for 10.000 and 5.000 starting cells was performed (Figure 3). The data show very high correlations with Pearson’s coefficients of 0.96 for both conditions.</p>
<div class="row">
<div class="small-6 columns" style="text-align: left;"><center>A. Auto ChIPmentation on 10.000 cells<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/auto-chipmentation-on-10000cells.jpg" caption="false" width="480" height="420" /></center></div>
<div class="small-6 columns"><center>B. Auto ChIPmentation on 5.000 cells<br /> <img src="https://www.diagenode.com/img/categories/kits_chromatin_function/auto-chipmentation-on-5000cells.jpg" width="480" height="420" /></center></div>
</div>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure3"><i class="fa fa-caret-right"></i> Figure 3</a>
<div class="content" id="figure3">
<p><strong>Correlation of replicates of auto-ChIPmentation experiments.</strong> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 10.000 and 5.000 cells was used for the immunoprecipitation with the Diagenode antibody targeting H3K4me3 (Cat. no. C15410003). The experiments were performed on the IP-Star® Compact Automated System.The scatterplots show the correlation between the enrichments between two replicates. The green line represents an ideal case where the two replicates are identical, while the red curve fitted on the data points by a LOESS regression shows the actual trend. A. Correlation analysis between two replicates of auto-ChIPmentation experiments starting from 10.000 cells. B. Correlation analysis between two replicates of auto-ChIPmentation experiments starting from 5.000 cells.</p>
</div>
</li>
</ul>
<p>The Auto-ChIPmentation workflow is easy and fast, producing high-quality ChIP-seq data even on low starting amounts.</p>
<center><img src="https://www.diagenode.com/img/product/chipmentation/chipmentation-sequencing-results-for-histone.jpg" width="678" height="240" /></center>
<ul class="accordion extra-spaced" data-accordion="">
<li class="accordion-navigation"><a href="#figure4"><i class="fa fa-caret-right"></i> Figure 4</a>
<div class="content" id="figure4">
<p><strong>ChIPmentation sequencing results for four histone marks.</strong> Chromatin preparation has been performed on 7 M K562 cells using the Auto ChIPmentation Kit for Histones (Cat. no. C01011000). Diluted chromatin from 1,000,000 cells was used for the immunoprecipitation with the Diagenode antibodies targeting H3K4me1 (Cat. no. C15410194), H3K27me3 (Cat. no. C15410195), H3K27ac (Cat. no. C15410196) and IgG (Cat. no. C15410206). The experiment was performed on the IP-Star® Compact Automated System.</p>
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