H3K36me3 Antibody
Polyclonal antibody raised in rabbit against histone H3, trimethylated at lysine 36 (H3K36me3), using a KLH-conjugated synthetic peptide.
| Lot | A8889-001P |
|---|---|
| Concentration | 0.9 µg/µl |
| Species reactivity | Human, mouse, zebrafish, Drosophila: positive. Other species: not tested. |
| Type | Polyclonal |
| Purity | Affinity purified |
| Host | Rabbit |
| 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. |
| Applications | Suggested dilution | References |
|---|---|---|
| ChIP/ChIP-seq * | 1-2 μg/ChIP | Fig 1, 2 |
| ELISA | 1:500 | Fig 3 |
| Dot Blotting | 1:20,000 | Fig 4 |
| Western Blotting | 1:1,000 | Fig 5 |
* Please note that the optimal antibody amount per IP should be determined by the end-user. We recommend testing 1-5 μg per IP.
Validation Data
Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K36me3
ChIP assays were performed using HeLa cells, the Diagenode antibody against H3K36me3 (cat. No. C15410058) and optimized PCR primer sets for qPCR. ChIP was performed with the “Auto Histone ChIP-seq” kit (cat. No. C01010022), using sheared chromatin from 1 million cells on the SX-8G IP-Star automated system. A titration consisting of 1, 2, 5 and 10 µg of antibody per ChIP experiment was analyzed. IgG (2 µg/IP) was used as a negative IP control. QPCR was performed with primers for the promoter and coding region of the active GAPDH, for a region located 1 kb upstream of the GAPDH promoter (cat. No. C17011003) and for the Sat2 satellite repeat. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).
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Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H3K36me3
ChIP was performed with 2 µg of the Diagenode antibody against H3K36me3 (cat. No. C15410058) on sheared chromatin from 1 million HeLaS3 cells using the “iDeal ChIP-seq” kit (cat. No. C01010051). IgG (2 µg/IP) was used as a negative IP control. The IP'd DNA was analysed by QPCR with optimized PCR primer pairs for the promoter and coding region of the active GAPDH, for a region located 1 kb upstream of the GAPDH promoter and for the coding region of the active ACTB gene (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 obtained profiles in genomic regions of chromosome 12 (including the GAPDH positive control), 7 (including the ACTB positive control), 14 and 3, respectively. These results clearly show an enrichment of the H3K36me3 at active genes.
Figure 3. Determination of the antibody titer
To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H3K36me3 (cat. No. C15410058) and the crude serum. 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 purified antibody was estimated to be 1:19,300.
Figure 4. Cross reactivity tests using the Diagenode antibody directed against H3K36me3
A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K36me3 (cat. No. C15410058) with peptides containing other H3 and H4 modifications and the unmodified sequence. One hundred to 0.2 pmol of peptide containing the respective histone modification were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 4 shows a high specificity of the antibody for the modification of interest.
Figure 5. Western blot analysis using the Diagenode antibody directed against H3K36me3
Histone extracts (15 µg) from HeLa cells were analysed by Western blot using the Diagenode antibody directed against H3K36me3 (cat. No. C15410058) 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.
Target 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 H3K36 is associated with actively transcribed regions.
Applications
| ELISA Enzyme-linked immunosorbent assay. Read more |
| DB Dot blotting Read more |
| WB Western blot : 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. Learn more about: Load... Read more |
| ChIP-seq (ab) Read more |
| ChIP-qPCR (ab) Read more |
Documents
| Datasheet H3K36me3 C15410058 DATASHEET Datasheet description | Download |
| Antibodies you can trust POSTER Epigenetic research tools have evolved over time from endpoint PCR to qPCR to the analyses of lar... | Download |
| Epigenetic Antibodies Brochure BROCHURE More than in any other immuoprecipitation assays, quality antibodies are critical tools in many e... | Download |
Safety sheets
Publications
How to properly cite our product/service in your workWe strongly recommend using this: H3K36me3 Antibody (Hologic Diagenode Cat# C15410058 Lot# A8889-001P). Click here to copy to clipboard. Using our products or services in your publication? Let us know! |
Chromatin profiling identifies transcriptional readthrough as a conservedmechanism for piRNA biogenesis in mosquitoes. |
Gene Regulatory Interactions at Lamina-Associated Domains |
Epigenetic Mechanisms Mediating Cell State Transitions in Chondrocytes |
p53 directly represses human LINE1 transposons. |
Premature termination codons in the gene cause reduced local mRNA synthesis. |
The Elongin Complex Antagonizes the Chromatin Factor Corto for Vein versus Intervein Cell Identity in Drosophila Wings. |
Differential transcript isoform usage pre- and post-zygotic genome activation in zebrafish. |
Regulation of transcription through acetylation of H3K122 on the lateral surface of the histone octamer. |
Prepatterning of developmental gene expression by modified histones before zygotic genome activation. |
Characterization of the contradictory chromatin signatures at the 3' exons of zinc finger genes. |
Promoter-exon relationship of H3 lysine 9, 27, 36 and 79 methylation on pluripotency-associated genes. |
Chromatin states of core pluripotency-associated genes in pluripotent, multipotent and differentiated cells. |