H3K36me3 polyclonal antibody - Premium
Polyclonal antibody raised in rabbit against the region of histone H3 containing the trimethylated lysine 36 (H3K36me3), using a KLH-conjugated synthetic peptide.
| Lot | A1845P |
|---|---|
| Concentration | 1.6 μg/μl |
| Species reactivity | Human, mouse, Arabidopsis, rice, wide range expected. |
| Type | Polyclonal |
| Purity | Affinity purified polyclonal antibody. |
|---|---|
| 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 * | 0.5 - 1 μg per IP | Fig 1, 2 |
| ELISA | 1:4,000 | Fig 3 |
| Dot Blotting/Peptide array | 1:20,000/1:10,000 | Fig 4 |
| Western Blotting | 1:1,000 | Fig 5 |
| Immunofluorescence | 1:500 | Fig 6 |
* 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.
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B01060010Bioruptor® Pico sonication device
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B03000002IP-Star® Compact Automated System
Validation Data
Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K36me3
Figure 1A ChIP assays were performed using human HeLa cells, the Diagenode antibody against H3K36me3 (Cat. No. C15410192) and optimized PCR primer pairs for qPCR. ChIP was performed with the “Auto Histone ChIP-seq” kit (Cat. No. C01010022) on the IP-Star automated system, using sheared chromatin from 1,000,000 cells. 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. Quantitative PCR was performed with primers for the coding region of the active GAPDH and ACTB genes, used as positive controls, and for the promoter and a region located 1 kb upstream of the promoter of the GAPDH gene, used as negative controls.
Figure 1B ChIP assays were performed using human K562 cells, the Diagenode antibody against H3K36me3 (Cat. No. C15410192) and optimized PCR primer pairs for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (Cat. No. C01010051), using sheared chromatin from 100,000 cells. 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. Quantitative PCR was performed with primers for the coding region of the active GAPDH and ACTB genes, used as positive controls, and for the coding region of the inactive MB gene and the Sat satellite repeat, used as negative controls. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).
Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H3K36me3
ChIP was performed on sheared chromatin from 100,000 K562 cells with the “iDeal ChIP-seq” kit (Cat. No. C01010051) using 0.5 μg of the Diagenode antibody against H3K36me3 (Cat. No. C15410192) as described above. 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 2 shows the H3K36me3 signal distribution along the complete sequence and a zoomin of human chromosome 12 (figure 2A and B) and in 2 genomic regions containing the GAPDH and ACTB positive control genes (figure 2C and D).
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 against H3K36me3 (Cat. No. C15410192). 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 1:132,000.
Figure 4. Cross reactivity tests using the Diagenode antibody directed against H3K36me3
Figure 4A. To test the cross reactivity of the Diagenode antibody against H3K36me3 (Cat. No. C15410192), a Dot Blot analysis was performed with peptides containing other modifications or unmodified sequences of histone H3 and H4. 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. The peptide array analysis shows a slight cross reaction with H4K20me3 that was not observed in dot blot.
Figure 5. Western blot analysis using the Diagenode antibody directed against H3K36me3
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 H3K36me3 (Cat. No. C15410192). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is shown on the right, the marker (in kDa) is shown on the left.
Figure 6. Immunofluorescence using the Diagenode antibody directed against H3K36me3
HeLa cells were stained with the Diagenode antibody against H3K36me3 (Cat. C15410192) and with DAPI. Cells were fixed with 4% formaldehyde for 10’ and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H3K36me3 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.
Testimonials
I am working with the True MicroChIP & Microplex Library Preparation Kits and several histone modification antibodies like H3K27ac, H3K4me3, H3K36me3, and H3K27me3. I got always very good and reproducible results for my ChIP-seq experiments.
Andrea Thiesen, ZMB, Developmental Biology, Prof. Dr. Andrea Vortkamp´s lab, University Duisburg-Essen, Germany
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 |
| IF Immunofluorescence: Diagenode offers huge selection of highly sensitive antibodies validated in IF. Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9 HeLa cells transfected with a Cas9 expression vector (... Read more |
| ChIP-seq (ab) Read more |
| ChIP-qPCR (ab) Read more |
Documents
| Datasheet H3K36me3 C15410192 DATASHEET Polyclonal antibody raised in rabbit against the region of histone H3 containing the trimethylate... | Download |
| Epigenetic Antibodies Brochure BROCHURE More than in any other immuoprecipitation assays, quality antibodies are critical tools in many e... | Download |
| Antibodies you can trust POSTER Epigenetic research tools have evolved over time from endpoint PCR to qPCR to the analyses of lar... | Download |
Publications
How to properly cite this product in your workDiagenode strongly recommends using this: H3K36me3 polyclonal antibody - Premium (Diagenode Cat# C15410192 Lot# A1845P). Click here to copy to clipboard. Using our products in your publication? Let us know! |
Reactivation of super-enhancers by KLF4 in human Head and Neck Squamous Cell Carcinoma. |
Chromatin-Based Classification of Genetically Heterogeneous AMLs into Two Distinct Subtypes with Diverse Stemness Phenotypes. |
Integrative Proteomic Profiling Reveals PRC2-Dependent Epigenetic Crosstalk Maintains Ground-State Pluripotency. |
The Itaconate Pathway Is a Central Regulatory Node Linking Innate Immune Tolerance and Trained Immunity |
The Polycomb-Dependent Epigenome Controls β Cell Dysfunction, Dedifferentiation, and Diabetes. |
The reference epigenome and regulatory chromatin landscape of chronic lymphocytic leukemia |
Increased H3K9 methylation and impaired expression of Protocadherins are associated with the cognitive dysfunctions of the Kleefstra syndrome. |
PRDM9 Methyltransferase Activity Is Essential for Meiotic DNA Double-Strand Break Formation at Its Binding Sites. |
Chromosome contacts in activated T cells identify autoimmune disease candidate genes |
Platelet function is modified by common sequence variation in megakaryocyte super enhancers |
DNA methylation heterogeneity defines a disease spectrum in Ewing sarcoma |
β-Glucan Reverses the Epigenetic State of LPS-Induced Immunological Tolerance |
The Hematopoietic Transcription Factors RUNX1 and ERG Prevent AML1-ETO Oncogene Overexpression and Onset of the Apoptosis Program in t(8;21) AMLs |
Neonatal monocytes exhibit a unique histone modification landscape |
Epigenetic dynamics of monocyte-to-macrophage differentiation |
Comprehensive genome and epigenome characterization of CHO cells in response to evolutionary pressures and over time |
Epigenome mapping reveals distinct modes of gene regulation and widespread enhancer reprogramming by the oncogenic fusion protein EWS-FLI1. |
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