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Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K64me3 ChIP assays were performed using human K562 cells, the Diagenode antibody against H3K64me3 (Cat. No. 15410211) and optimized PCR primer sets for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (Cat. No. C01010051) on sheared chromatin from 1 million cells. A titration of the antibody consisting of 1, 2, 5, and 10 μg per ChIP experiment was analysed. IgG (2 μg/IP) was used as negative IP control. QPCR was performed with primers for the promoter of the active GAPDH and EIF4A2 genes, used as negative controls, and for the Sat2 and Sata satellite repeats, used as positive 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. Determination of the antibody titer To determine the titer, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H3K64me3 (Cat. No. 15410211) in antigen coated wells. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the antibody was estimated to be 1:5,500.
Figure 3. Cross reactivity tests using the Diagenode antibody directed against H3K64me3 To check the specificity of the Diagenode antibody against H3K64me3 (Cat. No C15410211) a Dot Blot was performed with peptides containing different modifications of histone H3 and H4 or the unmodified H3K64 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 1:5,000. Figure 3 shows a high specificity of the antibody for the modification of interest.
Figure 4. Western blot analysis using the Diagenode antibody directed against H3K64me3 Western blot was performed on histone extracts (30 μg) from HeLa cells using the Diagenode antibody against H3K64me3 (Cat. No. C15410211). The antibody was diluted 1:100 in TBS-Tween containing 5% skimmed milk. The marker (in kDa) is shown on the left, the position of the protein is indicated on the right.
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.
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