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'description' => '<p><span>Polyclonal antibody raised in rabbit against histone H3 containing the trimethylated lysine 27 and the phosphorylated serine 28 (H3K27me3S28p), using a KLH-conjugated synthetic peptide.</span></p>',
'label1' => 'Validation Data',
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<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig6.png" alt="H3K27me3S28p Antibody ChIP Grade" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<div class="small-8 columns">
<p><small><strong> Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K27me3S28p </strong><br />ChIP assays were performed using HeLa cells treated with colcemid, the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) and optimized PCR primer pairs for qPCR. ChIP was performed with the “LowCell# ChIP” kit (cat. No. kch-maglow-016), using sheared chromatin from 10,000 cells. A titration of the antibody consisting of 1, 5 and 10 μl per ChIP experiment was analysed. Additionally, ChIP was performed after incubation of the antibody with 5 nmol blocking peptide (cat. No. sp-091-050) for 1 hour at room temperature. IgG (5 μg/IP) was used as negative IP control. QPCR was performed with primers for the promoters of the active genes c-fos (cat. No. pp-1004-050) and RPL30 and for the inactive gene MYOD. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). </small></p>
</div>
</div>
<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig1.png" alt="H3K27me3S28p Antibody ELISA validation" caption="false" width="288" height="229" /></p>
</div>
<div class="small-8 columns">
<p><small><strong> Figure 2. Determination of the titer </strong><br />To determine the titer, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H3K27me3S28p (cat. No. CS-091-100). The antigen used was a peptide containing the histone modifications of interest. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the antibody was estimated to be 1:8,300. </small></p>
</div>
</div>
<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig2.png" alt="H3K27me3S28p Antibody validated in Dot Blot" style="display: block; margin-left: auto; margin-right: auto;" /></p>
</div>
<div class="small-6 columns">
<p><small><strong> Figure 3. Cross reactivity test using the Diagenode antibody directed against H3K27me3S28p </strong><br />A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) with peptides containing other modifications of histone H3 and H4 and unmodified sequences from histone H3. One hundred to 0.2 pmol of the peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 3 shows a high specificity of the antibody for the peptide containing the modifications of interest. </small></p>
</div>
</div>
<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig3.png" alt="H3K27me3S28p Antibody validated in Western Blot" style="display: block; margin-left: auto; margin-right: auto;" /></p>
</div>
<div class="small-8 columns">
<p><small><strong> Figure 4. Western blot analysis using the Diagenode antibody directed against H3K27me3S28p </strong><br />Histone extracts of HeLa cells (15 μg) were analysed by Western blot using the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) diluted 1:250 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the left; the marker (in kDa) is shown on the right. Lane 2 shows the result of the Western analysis with the antibody; lane 1 shows the same analysis after incubation of the antibody with 750 pmol blocking peptide (cat. No. sp-091-050) for 1 hour at room temperature. </small></p>
</div>
</div>
<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig4.png" alt="H3K27me3S28p Antibody validated in Immunofluorescence" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<div class="small-8 columns">
<p><small><strong> Figure 5. Immunofluorescence with the Diagenode antibody directed against H3K27me3S28p </strong><br />Hela asynchronous cells were stained with the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) and with DAPI. Cells were fixed with formaldehyde, permeabilized with sodium citrate and Triton X100 and blocked with PBS containing 2.5% BSA. Figure 5A: cells were immunofluorescently labelled with the H3K27me3S28p antibody (diluted 1:200 and incubated for 1 hour at room temperature) followed by goat anti-rabbit antibody conjugated to DyLight 488. Figure 5B: the nuclei were stained with DAPI, which specifically labels DNA. Phosphorylation of H3 on serine 28 is increased during late G2 phase and reaches a maximum in metaphase cells. This may explain the different staining intensities of different cells. </small></p>
</div>
</div>
<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig5.png" alt="H3K27me3S28p Antibody validated in Immunoprecipitation" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<div class="small-8 columns">
<p><small><strong> Figure 6. Immunoprecipitation with the Diagenode antibody directed against H3K27me3S28p </strong><br />HeLa cells were treated with colcemid to block the cell cycle in metaphase and were fixed with formaldehyde. Chromatin from 10,000 cells was sheared and used for immunoprecipitation (IP). IP was performed with 5 μl of the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100). The immunoprecipitated proteins were analysed by Western blot with the antibody diluted 1:500 in TBS-Tween containing 5% skimmed milk. Lane 1 shows the result of the IP; a negative IP control (no antibody added) and a positive control (sheared chromatin from 10,000 cells) are shown in lane 2 and 3, respectively. </small></p>
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<td>ChIP <sup>*</sup> </td>
<td>1 μl/ChIP</td>
<td>Fig 1</td>
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<tr>
<td>ELISA</td>
<td>1:100 - 1:500</td>
<td>Fig 2</td>
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<tr>
<td>Dot Blotting</td>
<td>1:20,000</td>
<td>Fig 3</td>
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<td>Western Blotting</td>
<td>1:250 - 1:500</td>
<td>Fig 4, Fig 6</td>
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<tr>
<td>Immunofluorescence</td>
<td>1:200</td>
<td>Fig 5</td>
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<td>Immunoprecipitation</td>
<td>5 μl/IP</td>
<td>Fig 6</td>
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<small><sup>*</sup>
Please note that of the optimal antibody amount per IP should be determined by the end-user. We recommend testing 1-10 μl per IP.</small>',
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$meta_description = 'H3K27me3S28p (Histone H3 trimethylated at lysine 27 and phosphorylated at serine 28) Polyclonal Antibody validated in ChIP-qPCR, ELISA, DB, WB, IF and IP. Batch-specific data available on the website. '
$meta_title = 'H3K27me3S28p Polyclonal Antibody | Diagenode'
$product = array(
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'name' => 'H3K27me3S28p polyclonal antibody ',
'description' => '<p><span>Polyclonal antibody raised in rabbit against histone H3 containing the trimethylated lysine 27 and the phosphorylated serine 28 (H3K27me3S28p), using a KLH-conjugated synthetic peptide.</span></p>',
'label1' => 'Validation Data',
'info1' => '<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig6.png" alt="H3K27me3S28p Antibody ChIP Grade" style="display: block; margin-left: auto; margin-right: auto;" /></p>
</div>
<div class="small-8 columns">
<p><small><strong> Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K27me3S28p </strong><br />ChIP assays were performed using HeLa cells treated with colcemid, the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) and optimized PCR primer pairs for qPCR. ChIP was performed with the “LowCell# ChIP” kit (cat. No. kch-maglow-016), using sheared chromatin from 10,000 cells. A titration of the antibody consisting of 1, 5 and 10 μl per ChIP experiment was analysed. Additionally, ChIP was performed after incubation of the antibody with 5 nmol blocking peptide (cat. No. sp-091-050) for 1 hour at room temperature. IgG (5 μg/IP) was used as negative IP control. QPCR was performed with primers for the promoters of the active genes c-fos (cat. No. pp-1004-050) and RPL30 and for the inactive gene MYOD. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). </small></p>
</div>
</div>
<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig1.png" alt="H3K27me3S28p Antibody ELISA validation" caption="false" width="288" height="229" /></p>
</div>
<div class="small-8 columns">
<p><small><strong> Figure 2. Determination of the titer </strong><br />To determine the titer, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H3K27me3S28p (cat. No. CS-091-100). The antigen used was a peptide containing the histone modifications of interest. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the antibody was estimated to be 1:8,300. </small></p>
</div>
</div>
<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig2.png" alt="H3K27me3S28p Antibody validated in Dot Blot" style="display: block; margin-left: auto; margin-right: auto;" /></p>
</div>
<div class="small-6 columns">
<p><small><strong> Figure 3. Cross reactivity test using the Diagenode antibody directed against H3K27me3S28p </strong><br />A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) with peptides containing other modifications of histone H3 and H4 and unmodified sequences from histone H3. One hundred to 0.2 pmol of the peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 3 shows a high specificity of the antibody for the peptide containing the modifications of interest. </small></p>
</div>
</div>
<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig3.png" alt="H3K27me3S28p Antibody validated in Western Blot" style="display: block; margin-left: auto; margin-right: auto;" /></p>
</div>
<div class="small-8 columns">
<p><small><strong> Figure 4. Western blot analysis using the Diagenode antibody directed against H3K27me3S28p </strong><br />Histone extracts of HeLa cells (15 μg) were analysed by Western blot using the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) diluted 1:250 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the left; the marker (in kDa) is shown on the right. Lane 2 shows the result of the Western analysis with the antibody; lane 1 shows the same analysis after incubation of the antibody with 750 pmol blocking peptide (cat. No. sp-091-050) for 1 hour at room temperature. </small></p>
</div>
</div>
<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig4.png" alt="H3K27me3S28p Antibody validated in Immunofluorescence" style="display: block; margin-left: auto; margin-right: auto;" /></p>
</div>
<div class="small-8 columns">
<p><small><strong> Figure 5. Immunofluorescence with the Diagenode antibody directed against H3K27me3S28p </strong><br />Hela asynchronous cells were stained with the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) and with DAPI. Cells were fixed with formaldehyde, permeabilized with sodium citrate and Triton X100 and blocked with PBS containing 2.5% BSA. Figure 5A: cells were immunofluorescently labelled with the H3K27me3S28p antibody (diluted 1:200 and incubated for 1 hour at room temperature) followed by goat anti-rabbit antibody conjugated to DyLight 488. Figure 5B: the nuclei were stained with DAPI, which specifically labels DNA. Phosphorylation of H3 on serine 28 is increased during late G2 phase and reaches a maximum in metaphase cells. This may explain the different staining intensities of different cells. </small></p>
</div>
</div>
<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig5.png" alt="H3K27me3S28p Antibody validated in Immunoprecipitation" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<div class="small-8 columns">
<p><small><strong> Figure 6. Immunoprecipitation with the Diagenode antibody directed against H3K27me3S28p </strong><br />HeLa cells were treated with colcemid to block the cell cycle in metaphase and were fixed with formaldehyde. Chromatin from 10,000 cells was sheared and used for immunoprecipitation (IP). IP was performed with 5 μl of the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100). The immunoprecipitated proteins were analysed by Western blot with the antibody diluted 1:500 in TBS-Tween containing 5% skimmed milk. Lane 1 shows the result of the IP; a negative IP control (no antibody added) and a positive control (sheared chromatin from 10,000 cells) are shown in lane 2 and 3, respectively. </small></p>
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<th>References</th>
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<td>ChIP <sup>*</sup> </td>
<td>1 μl/ChIP</td>
<td>Fig 1</td>
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<tr>
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<td>1:100 - 1:500</td>
<td>Fig 2</td>
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<td>1:20,000</td>
<td>Fig 3</td>
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<tr>
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<td>1:250 - 1:500</td>
<td>Fig 4, Fig 6</td>
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<tr>
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<td>1:200</td>
<td>Fig 5</td>
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<td>5 μl/IP</td>
<td>Fig 6</td>
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<small><sup>*</sup>
Please note that of the optimal antibody amount per IP should be determined by the end-user. We recommend testing 1-10 μl per IP.</small>',
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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><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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<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>
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<p><img src="https://www.diagenode.com/img/categories/antibodies/histone-marks-illustration.png" /></p>
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<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>
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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>
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<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><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-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig6.png" alt="H3K27me3S28p Antibody ChIP Grade" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<p><small><strong> Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K27me3S28p </strong><br />ChIP assays were performed using HeLa cells treated with colcemid, the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) and optimized PCR primer pairs for qPCR. ChIP was performed with the “LowCell# ChIP” kit (cat. No. kch-maglow-016), using sheared chromatin from 10,000 cells. A titration of the antibody consisting of 1, 5 and 10 μl per ChIP experiment was analysed. Additionally, ChIP was performed after incubation of the antibody with 5 nmol blocking peptide (cat. No. sp-091-050) for 1 hour at room temperature. IgG (5 μg/IP) was used as negative IP control. QPCR was performed with primers for the promoters of the active genes c-fos (cat. No. pp-1004-050) and RPL30 and for the inactive gene MYOD. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). </small></p>
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<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig1.png" alt="H3K27me3S28p Antibody ELISA validation" caption="false" width="288" height="229" /></p>
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<div class="small-8 columns">
<p><small><strong> Figure 2. Determination of the titer </strong><br />To determine the titer, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H3K27me3S28p (cat. No. CS-091-100). The antigen used was a peptide containing the histone modifications of interest. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the antibody was estimated to be 1:8,300. </small></p>
</div>
</div>
<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig2.png" alt="H3K27me3S28p Antibody validated in Dot Blot" style="display: block; margin-left: auto; margin-right: auto;" /></p>
</div>
<div class="small-6 columns">
<p><small><strong> Figure 3. Cross reactivity test using the Diagenode antibody directed against H3K27me3S28p </strong><br />A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) with peptides containing other modifications of histone H3 and H4 and unmodified sequences from histone H3. One hundred to 0.2 pmol of the peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 3 shows a high specificity of the antibody for the peptide containing the modifications of interest. </small></p>
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</div>
<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig3.png" alt="H3K27me3S28p Antibody validated in Western Blot" style="display: block; margin-left: auto; margin-right: auto;" /></p>
</div>
<div class="small-8 columns">
<p><small><strong> Figure 4. Western blot analysis using the Diagenode antibody directed against H3K27me3S28p </strong><br />Histone extracts of HeLa cells (15 μg) were analysed by Western blot using the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) diluted 1:250 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the left; the marker (in kDa) is shown on the right. Lane 2 shows the result of the Western analysis with the antibody; lane 1 shows the same analysis after incubation of the antibody with 750 pmol blocking peptide (cat. No. sp-091-050) for 1 hour at room temperature. </small></p>
</div>
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<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig4.png" alt="H3K27me3S28p Antibody validated in Immunofluorescence" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<div class="small-8 columns">
<p><small><strong> Figure 5. Immunofluorescence with the Diagenode antibody directed against H3K27me3S28p </strong><br />Hela asynchronous cells were stained with the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) and with DAPI. Cells were fixed with formaldehyde, permeabilized with sodium citrate and Triton X100 and blocked with PBS containing 2.5% BSA. Figure 5A: cells were immunofluorescently labelled with the H3K27me3S28p antibody (diluted 1:200 and incubated for 1 hour at room temperature) followed by goat anti-rabbit antibody conjugated to DyLight 488. Figure 5B: the nuclei were stained with DAPI, which specifically labels DNA. Phosphorylation of H3 on serine 28 is increased during late G2 phase and reaches a maximum in metaphase cells. This may explain the different staining intensities of different cells. </small></p>
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<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig5.png" alt="H3K27me3S28p Antibody validated in Immunoprecipitation" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<p><small><strong> Figure 6. Immunoprecipitation with the Diagenode antibody directed against H3K27me3S28p </strong><br />HeLa cells were treated with colcemid to block the cell cycle in metaphase and were fixed with formaldehyde. Chromatin from 10,000 cells was sheared and used for immunoprecipitation (IP). IP was performed with 5 μl of the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100). The immunoprecipitated proteins were analysed by Western blot with the antibody diluted 1:500 in TBS-Tween containing 5% skimmed milk. Lane 1 shows the result of the IP; a negative IP control (no antibody added) and a positive control (sheared chromatin from 10,000 cells) are shown in lane 2 and 3, respectively. </small></p>
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<td>1 μl/ChIP</td>
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<td>1:100 - 1:500</td>
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<td>1:20,000</td>
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<td>1:250 - 1:500</td>
<td>Fig 4, Fig 6</td>
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<td>1:200</td>
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<td>5 μl/IP</td>
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Please note that of the optimal antibody amount per IP should be determined by the end-user. We recommend testing 1-10 μl per IP.</small>',
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$meta_description = 'H3K27me3S28p (Histone H3 trimethylated at lysine 27 and phosphorylated at serine 28) Polyclonal Antibody validated in ChIP-qPCR, ELISA, DB, WB, IF and IP. Batch-specific data available on the website. '
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'description' => '<p><span>Polyclonal antibody raised in rabbit against histone H3 containing the trimethylated lysine 27 and the phosphorylated serine 28 (H3K27me3S28p), using a KLH-conjugated synthetic peptide.</span></p>',
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<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig6.png" alt="H3K27me3S28p Antibody ChIP Grade" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<p><small><strong> Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K27me3S28p </strong><br />ChIP assays were performed using HeLa cells treated with colcemid, the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) and optimized PCR primer pairs for qPCR. ChIP was performed with the “LowCell# ChIP” kit (cat. No. kch-maglow-016), using sheared chromatin from 10,000 cells. A titration of the antibody consisting of 1, 5 and 10 μl per ChIP experiment was analysed. Additionally, ChIP was performed after incubation of the antibody with 5 nmol blocking peptide (cat. No. sp-091-050) for 1 hour at room temperature. IgG (5 μg/IP) was used as negative IP control. QPCR was performed with primers for the promoters of the active genes c-fos (cat. No. pp-1004-050) and RPL30 and for the inactive gene MYOD. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). </small></p>
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<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig1.png" alt="H3K27me3S28p Antibody ELISA validation" caption="false" width="288" height="229" /></p>
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<p><small><strong> Figure 2. Determination of the titer </strong><br />To determine the titer, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H3K27me3S28p (cat. No. CS-091-100). The antigen used was a peptide containing the histone modifications of interest. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the antibody was estimated to be 1:8,300. </small></p>
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<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig2.png" alt="H3K27me3S28p Antibody validated in Dot Blot" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<p><small><strong> Figure 3. Cross reactivity test using the Diagenode antibody directed against H3K27me3S28p </strong><br />A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) with peptides containing other modifications of histone H3 and H4 and unmodified sequences from histone H3. One hundred to 0.2 pmol of the peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 3 shows a high specificity of the antibody for the peptide containing the modifications of interest. </small></p>
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<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig3.png" alt="H3K27me3S28p Antibody validated in Western Blot" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<div class="small-8 columns">
<p><small><strong> Figure 4. Western blot analysis using the Diagenode antibody directed against H3K27me3S28p </strong><br />Histone extracts of HeLa cells (15 μg) were analysed by Western blot using the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) diluted 1:250 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the left; the marker (in kDa) is shown on the right. Lane 2 shows the result of the Western analysis with the antibody; lane 1 shows the same analysis after incubation of the antibody with 750 pmol blocking peptide (cat. No. sp-091-050) for 1 hour at room temperature. </small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig4.png" alt="H3K27me3S28p Antibody validated in Immunofluorescence" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<p><small><strong> Figure 5. Immunofluorescence with the Diagenode antibody directed against H3K27me3S28p </strong><br />Hela asynchronous cells were stained with the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) and with DAPI. Cells were fixed with formaldehyde, permeabilized with sodium citrate and Triton X100 and blocked with PBS containing 2.5% BSA. Figure 5A: cells were immunofluorescently labelled with the H3K27me3S28p antibody (diluted 1:200 and incubated for 1 hour at room temperature) followed by goat anti-rabbit antibody conjugated to DyLight 488. Figure 5B: the nuclei were stained with DAPI, which specifically labels DNA. Phosphorylation of H3 on serine 28 is increased during late G2 phase and reaches a maximum in metaphase cells. This may explain the different staining intensities of different cells. </small></p>
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<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig5.png" alt="H3K27me3S28p Antibody validated in Immunoprecipitation" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<p><small><strong> Figure 6. Immunoprecipitation with the Diagenode antibody directed against H3K27me3S28p </strong><br />HeLa cells were treated with colcemid to block the cell cycle in metaphase and were fixed with formaldehyde. Chromatin from 10,000 cells was sheared and used for immunoprecipitation (IP). IP was performed with 5 μl of the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100). The immunoprecipitated proteins were analysed by Western blot with the antibody diluted 1:500 in TBS-Tween containing 5% skimmed milk. Lane 1 shows the result of the IP; a negative IP control (no antibody added) and a positive control (sheared chromatin from 10,000 cells) are shown in lane 2 and 3, respectively. </small></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><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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<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>
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<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>
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<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>
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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>
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<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><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><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig6.png" alt="H3K27me3S28p Antibody ChIP Grade" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<p><small><strong> Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K27me3S28p </strong><br />ChIP assays were performed using HeLa cells treated with colcemid, the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) and optimized PCR primer pairs for qPCR. ChIP was performed with the “LowCell# ChIP” kit (cat. No. kch-maglow-016), using sheared chromatin from 10,000 cells. A titration of the antibody consisting of 1, 5 and 10 μl per ChIP experiment was analysed. Additionally, ChIP was performed after incubation of the antibody with 5 nmol blocking peptide (cat. No. sp-091-050) for 1 hour at room temperature. IgG (5 μg/IP) was used as negative IP control. QPCR was performed with primers for the promoters of the active genes c-fos (cat. No. pp-1004-050) and RPL30 and for the inactive gene MYOD. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). </small></p>
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<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig1.png" alt="H3K27me3S28p Antibody ELISA validation" caption="false" width="288" height="229" /></p>
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<div class="small-8 columns">
<p><small><strong> Figure 2. Determination of the titer </strong><br />To determine the titer, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H3K27me3S28p (cat. No. CS-091-100). The antigen used was a peptide containing the histone modifications of interest. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the antibody was estimated to be 1:8,300. </small></p>
</div>
</div>
<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig2.png" alt="H3K27me3S28p Antibody validated in Dot Blot" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<div class="small-6 columns">
<p><small><strong> Figure 3. Cross reactivity test using the Diagenode antibody directed against H3K27me3S28p </strong><br />A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) with peptides containing other modifications of histone H3 and H4 and unmodified sequences from histone H3. One hundred to 0.2 pmol of the peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 3 shows a high specificity of the antibody for the peptide containing the modifications of interest. </small></p>
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<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig3.png" alt="H3K27me3S28p Antibody validated in Western Blot" style="display: block; margin-left: auto; margin-right: auto;" /></p>
</div>
<div class="small-8 columns">
<p><small><strong> Figure 4. Western blot analysis using the Diagenode antibody directed against H3K27me3S28p </strong><br />Histone extracts of HeLa cells (15 μg) were analysed by Western blot using the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) diluted 1:250 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the left; the marker (in kDa) is shown on the right. Lane 2 shows the result of the Western analysis with the antibody; lane 1 shows the same analysis after incubation of the antibody with 750 pmol blocking peptide (cat. No. sp-091-050) for 1 hour at room temperature. </small></p>
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<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig4.png" alt="H3K27me3S28p Antibody validated in Immunofluorescence" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<div class="small-8 columns">
<p><small><strong> Figure 5. Immunofluorescence with the Diagenode antibody directed against H3K27me3S28p </strong><br />Hela asynchronous cells were stained with the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) and with DAPI. Cells were fixed with formaldehyde, permeabilized with sodium citrate and Triton X100 and blocked with PBS containing 2.5% BSA. Figure 5A: cells were immunofluorescently labelled with the H3K27me3S28p antibody (diluted 1:200 and incubated for 1 hour at room temperature) followed by goat anti-rabbit antibody conjugated to DyLight 488. Figure 5B: the nuclei were stained with DAPI, which specifically labels DNA. Phosphorylation of H3 on serine 28 is increased during late G2 phase and reaches a maximum in metaphase cells. This may explain the different staining intensities of different cells. </small></p>
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</div>
<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig5.png" alt="H3K27me3S28p Antibody validated in Immunoprecipitation" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<div class="small-8 columns">
<p><small><strong> Figure 6. Immunoprecipitation with the Diagenode antibody directed against H3K27me3S28p </strong><br />HeLa cells were treated with colcemid to block the cell cycle in metaphase and were fixed with formaldehyde. Chromatin from 10,000 cells was sheared and used for immunoprecipitation (IP). IP was performed with 5 μl of the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100). The immunoprecipitated proteins were analysed by Western blot with the antibody diluted 1:500 in TBS-Tween containing 5% skimmed milk. Lane 1 shows the result of the IP; a negative IP control (no antibody added) and a positive control (sheared chromatin from 10,000 cells) are shown in lane 2 and 3, respectively. </small></p>
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<td>1 μl/ChIP</td>
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Please note that of the optimal antibody amount per IP should be determined by the end-user. We recommend testing 1-10 μl per IP.</small>',
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<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig6.png" alt="H3K27me3S28p Antibody ChIP Grade" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<p><small><strong> Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K27me3S28p </strong><br />ChIP assays were performed using HeLa cells treated with colcemid, the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) and optimized PCR primer pairs for qPCR. ChIP was performed with the “LowCell# ChIP” kit (cat. No. kch-maglow-016), using sheared chromatin from 10,000 cells. A titration of the antibody consisting of 1, 5 and 10 μl per ChIP experiment was analysed. Additionally, ChIP was performed after incubation of the antibody with 5 nmol blocking peptide (cat. No. sp-091-050) for 1 hour at room temperature. IgG (5 μg/IP) was used as negative IP control. QPCR was performed with primers for the promoters of the active genes c-fos (cat. No. pp-1004-050) and RPL30 and for the inactive gene MYOD. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). </small></p>
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<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig1.png" alt="H3K27me3S28p Antibody ELISA validation" caption="false" width="288" height="229" /></p>
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<p><small><strong> Figure 2. Determination of the titer </strong><br />To determine the titer, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H3K27me3S28p (cat. No. CS-091-100). The antigen used was a peptide containing the histone modifications of interest. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the antibody was estimated to be 1:8,300. </small></p>
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<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig2.png" alt="H3K27me3S28p Antibody validated in Dot Blot" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<p><small><strong> Figure 3. Cross reactivity test using the Diagenode antibody directed against H3K27me3S28p </strong><br />A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) with peptides containing other modifications of histone H3 and H4 and unmodified sequences from histone H3. One hundred to 0.2 pmol of the peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 3 shows a high specificity of the antibody for the peptide containing the modifications of interest. </small></p>
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<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig3.png" alt="H3K27me3S28p Antibody validated in Western Blot" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<div class="small-8 columns">
<p><small><strong> Figure 4. Western blot analysis using the Diagenode antibody directed against H3K27me3S28p </strong><br />Histone extracts of HeLa cells (15 μg) were analysed by Western blot using the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) diluted 1:250 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the left; the marker (in kDa) is shown on the right. Lane 2 shows the result of the Western analysis with the antibody; lane 1 shows the same analysis after incubation of the antibody with 750 pmol blocking peptide (cat. No. sp-091-050) for 1 hour at room temperature. </small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig4.png" alt="H3K27me3S28p Antibody validated in Immunofluorescence" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<p><small><strong> Figure 5. Immunofluorescence with the Diagenode antibody directed against H3K27me3S28p </strong><br />Hela asynchronous cells were stained with the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) and with DAPI. Cells were fixed with formaldehyde, permeabilized with sodium citrate and Triton X100 and blocked with PBS containing 2.5% BSA. Figure 5A: cells were immunofluorescently labelled with the H3K27me3S28p antibody (diluted 1:200 and incubated for 1 hour at room temperature) followed by goat anti-rabbit antibody conjugated to DyLight 488. Figure 5B: the nuclei were stained with DAPI, which specifically labels DNA. Phosphorylation of H3 on serine 28 is increased during late G2 phase and reaches a maximum in metaphase cells. This may explain the different staining intensities of different cells. </small></p>
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<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig5.png" alt="H3K27me3S28p Antibody validated in Immunoprecipitation" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<p><small><strong> Figure 6. Immunoprecipitation with the Diagenode antibody directed against H3K27me3S28p </strong><br />HeLa cells were treated with colcemid to block the cell cycle in metaphase and were fixed with formaldehyde. Chromatin from 10,000 cells was sheared and used for immunoprecipitation (IP). IP was performed with 5 μl of the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100). The immunoprecipitated proteins were analysed by Western blot with the antibody diluted 1:500 in TBS-Tween containing 5% skimmed milk. Lane 1 shows the result of the IP; a negative IP control (no antibody added) and a positive control (sheared chromatin from 10,000 cells) are shown in lane 2 and 3, respectively. </small></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>
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<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>
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<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>
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<li><span style="font-weight: 400;"> Highly sensitive and specific</span></li>
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<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><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><small><strong> Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K27me3S28p </strong><br />ChIP assays were performed using HeLa cells treated with colcemid, the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) and optimized PCR primer pairs for qPCR. ChIP was performed with the “LowCell# ChIP” kit (cat. No. kch-maglow-016), using sheared chromatin from 10,000 cells. A titration of the antibody consisting of 1, 5 and 10 μl per ChIP experiment was analysed. Additionally, ChIP was performed after incubation of the antibody with 5 nmol blocking peptide (cat. No. sp-091-050) for 1 hour at room temperature. IgG (5 μg/IP) was used as negative IP control. QPCR was performed with primers for the promoters of the active genes c-fos (cat. No. pp-1004-050) and RPL30 and for the inactive gene MYOD. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). </small></p>
</div>
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<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig1.png" alt="H3K27me3S28p Antibody ELISA validation" caption="false" width="288" height="229" /></p>
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<div class="small-8 columns">
<p><small><strong> Figure 2. Determination of the titer </strong><br />To determine the titer, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H3K27me3S28p (cat. No. CS-091-100). The antigen used was a peptide containing the histone modifications of interest. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the antibody was estimated to be 1:8,300. </small></p>
</div>
</div>
<div class="row">
<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig2.png" alt="H3K27me3S28p Antibody validated in Dot Blot" style="display: block; margin-left: auto; margin-right: auto;" /></p>
</div>
<div class="small-6 columns">
<p><small><strong> Figure 3. Cross reactivity test using the Diagenode antibody directed against H3K27me3S28p </strong><br />A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) with peptides containing other modifications of histone H3 and H4 and unmodified sequences from histone H3. One hundred to 0.2 pmol of the peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 3 shows a high specificity of the antibody for the peptide containing the modifications of interest. </small></p>
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<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig3.png" alt="H3K27me3S28p Antibody validated in Western Blot" style="display: block; margin-left: auto; margin-right: auto;" /></p>
</div>
<div class="small-8 columns">
<p><small><strong> Figure 4. Western blot analysis using the Diagenode antibody directed against H3K27me3S28p </strong><br />Histone extracts of HeLa cells (15 μg) were analysed by Western blot using the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) diluted 1:250 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the left; the marker (in kDa) is shown on the right. Lane 2 shows the result of the Western analysis with the antibody; lane 1 shows the same analysis after incubation of the antibody with 750 pmol blocking peptide (cat. No. sp-091-050) for 1 hour at room temperature. </small></p>
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<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig4.png" alt="H3K27me3S28p Antibody validated in Immunofluorescence" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<div class="small-8 columns">
<p><small><strong> Figure 5. Immunofluorescence with the Diagenode antibody directed against H3K27me3S28p </strong><br />Hela asynchronous cells were stained with the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) and with DAPI. Cells were fixed with formaldehyde, permeabilized with sodium citrate and Triton X100 and blocked with PBS containing 2.5% BSA. Figure 5A: cells were immunofluorescently labelled with the H3K27me3S28p antibody (diluted 1:200 and incubated for 1 hour at room temperature) followed by goat anti-rabbit antibody conjugated to DyLight 488. Figure 5B: the nuclei were stained with DAPI, which specifically labels DNA. Phosphorylation of H3 on serine 28 is increased during late G2 phase and reaches a maximum in metaphase cells. This may explain the different staining intensities of different cells. </small></p>
</div>
</div>
<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig5.png" alt="H3K27me3S28p Antibody validated in Immunoprecipitation" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<div class="small-8 columns">
<p><small><strong> Figure 6. Immunoprecipitation with the Diagenode antibody directed against H3K27me3S28p </strong><br />HeLa cells were treated with colcemid to block the cell cycle in metaphase and were fixed with formaldehyde. Chromatin from 10,000 cells was sheared and used for immunoprecipitation (IP). IP was performed with 5 μl of the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100). The immunoprecipitated proteins were analysed by Western blot with the antibody diluted 1:500 in TBS-Tween containing 5% skimmed milk. Lane 1 shows the result of the IP; a negative IP control (no antibody added) and a positive control (sheared chromatin from 10,000 cells) are shown in lane 2 and 3, respectively. </small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig6.png" alt="H3K27me3S28p Antibody ChIP Grade" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<p><small><strong> Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K27me3S28p </strong><br />ChIP assays were performed using HeLa cells treated with colcemid, the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) and optimized PCR primer pairs for qPCR. ChIP was performed with the “LowCell# ChIP” kit (cat. No. kch-maglow-016), using sheared chromatin from 10,000 cells. A titration of the antibody consisting of 1, 5 and 10 μl per ChIP experiment was analysed. Additionally, ChIP was performed after incubation of the antibody with 5 nmol blocking peptide (cat. No. sp-091-050) for 1 hour at room temperature. IgG (5 μg/IP) was used as negative IP control. QPCR was performed with primers for the promoters of the active genes c-fos (cat. No. pp-1004-050) and RPL30 and for the inactive gene MYOD. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). </small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig1.png" alt="H3K27me3S28p Antibody ELISA validation" caption="false" width="288" height="229" /></p>
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<p><small><strong> Figure 2. Determination of the titer </strong><br />To determine the titer, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H3K27me3S28p (cat. No. CS-091-100). The antigen used was a peptide containing the histone modifications of interest. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the antibody was estimated to be 1:8,300. </small></p>
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<div class="small-6 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig2.png" alt="H3K27me3S28p Antibody validated in Dot Blot" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<p><small><strong> Figure 3. Cross reactivity test using the Diagenode antibody directed against H3K27me3S28p </strong><br />A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) with peptides containing other modifications of histone H3 and H4 and unmodified sequences from histone H3. One hundred to 0.2 pmol of the peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 3 shows a high specificity of the antibody for the peptide containing the modifications of interest. </small></p>
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<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig3.png" alt="H3K27me3S28p Antibody validated in Western Blot" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<div class="small-8 columns">
<p><small><strong> Figure 4. Western blot analysis using the Diagenode antibody directed against H3K27me3S28p </strong><br />Histone extracts of HeLa cells (15 μg) were analysed by Western blot using the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) diluted 1:250 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the left; the marker (in kDa) is shown on the right. Lane 2 shows the result of the Western analysis with the antibody; lane 1 shows the same analysis after incubation of the antibody with 750 pmol blocking peptide (cat. No. sp-091-050) for 1 hour at room temperature. </small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig4.png" alt="H3K27me3S28p Antibody validated in Immunofluorescence" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<p><small><strong> Figure 5. Immunofluorescence with the Diagenode antibody directed against H3K27me3S28p </strong><br />Hela asynchronous cells were stained with the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) and with DAPI. Cells were fixed with formaldehyde, permeabilized with sodium citrate and Triton X100 and blocked with PBS containing 2.5% BSA. Figure 5A: cells were immunofluorescently labelled with the H3K27me3S28p antibody (diluted 1:200 and incubated for 1 hour at room temperature) followed by goat anti-rabbit antibody conjugated to DyLight 488. Figure 5B: the nuclei were stained with DAPI, which specifically labels DNA. Phosphorylation of H3 on serine 28 is increased during late G2 phase and reaches a maximum in metaphase cells. This may explain the different staining intensities of different cells. </small></p>
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<div class="row">
<div class="small-4 columns">
<p><img src="https://www.diagenode.com/img/product/antibodies/C15310091_fig5.png" alt="H3K27me3S28p Antibody validated in Immunoprecipitation" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<p><small><strong> Figure 6. Immunoprecipitation with the Diagenode antibody directed against H3K27me3S28p </strong><br />HeLa cells were treated with colcemid to block the cell cycle in metaphase and were fixed with formaldehyde. Chromatin from 10,000 cells was sheared and used for immunoprecipitation (IP). IP was performed with 5 μl of the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100). The immunoprecipitated proteins were analysed by Western blot with the antibody diluted 1:500 in TBS-Tween containing 5% skimmed milk. Lane 1 shows the result of the IP; a negative IP control (no antibody added) and a positive control (sheared chromatin from 10,000 cells) are shown in lane 2 and 3, respectively. </small></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>
<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>
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<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>
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<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><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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