H3K9ac Antibody - ChIP-seq Grade

Figure 1 ChIP-seq results obtained with the Diagenode antibody directed against H3K9ac
ChIP was performed as described above using 1 μg of the Diagenode antibody against H3K9ac (cat. No. pAb- 177-050). The IP’d DNA was subsequently analysed with 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 peak distribution along the complete sequence and a 800 kb region of the X-chromosome (figure 2A and B) and in 100 kb regions surrounding the GAPDH and c-fos genes (figure 2C and D). These results clearly show an enrichment of H3K9ac at the promoters of active genes.

Figure 2 ChIP results obtained with the Diagenode antibody directed against RARA
ChIP assays were performed using HeLa cells, the Diagenode antibody against H3K9ac (cat. No. pAb-177-050) and optimized PCR primer sets for qPCR. ChIP was performed with the “Auto Histone ChIP-seq” kit (cat. No. AB- Auto02-A100), using 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. Quantitative PCR was performed with primers for the promoter of the active c-fos (cat. No. pp-1004-050) and GAPDH genes, used as positive controls, and for the coding region of the inactive MB gene (cat. No. pp-1006-050) and the Sat2 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). These results are in accordance with the observation that H3K9 acetylation is enriched at the promoters of 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 H3K9ac (cat. No. pAb-177-050) in antigen coated wells. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the antibody was estimated to be 1:59,600.

Figure 4 Cross reactivity test using the Diagenode antibody directed against H3K9ac
A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K9ac (cat. No. pAb-177-050) with peptides containing other histone modifications and the unmodified H3K9 sequence. 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 4 shows a high specificity of the antibody for the modification of interest. Please note that this antibody recognizes the H3K9 acetylation, both in the presence and the absence of the K14 acetylation or the S10 phosphorylation.

Figure 5 Western blot analysis using the Diagenode antibody directed against RARA
Histone extracts of HeLa cells (15 μg) were analysed by Western blot using the Diagenode antibody against H3K9ac (cat. No. pAb-177-050) 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.