ChIP was performed with 1 µg ab232952 against Histone H3 (acetyl K9 + K14) on sheared chromatin from 1 million HeLa S3 cells. 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 promoters of the active GAPDH and c-fos genes, used as positive control targets, and the coding region of the inactive MB gene and the Sat2 satellite repeat, used as negative control targets. 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. The figure shows the peak distribution along the complete sequence and a 800 kb region of the X-chromosome (A and B) and in 100 kb regions surrounding the RBM3, GAPDH and c-fos genes (C, D and E). These results clearly show an enrichment of the H3K9/14 double acetylation at the promoters of active genes.

ChIP assays were performed using HeLa (human epithelial cell line from cervix adenocarcinoma) cells, ab232952 against Histone H3 (acetyl K9 + K14) and optimized primer pairs for qPCR. ChIP was performed using sheared chromatin from 1.5 million cells. A titration of ab232952 consisting of 1, 2, 5 and 10 µg per ChIP experiment was analysed. IgG (5 µg/IP) was used as negative IP control. QPCR was performed using primers specific for the promoter of the ACTB gene as a positive control target and for exon 2 of the MB gene as a negative control target. The figure shows the recovery (the relative amount of immunoprecipitated DNA compared to input DNA). These results confirm the observation that acetylation of H3K9/14 is present at active promoters.

ChIP was performed with 1 µg ab232952 against Histone H3 (acetyl K9 + K14) on sheared chromatin from 1 million HeLa S3 cells. 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 promoters of the active GAPDH and c-fos genes, used as positive control targets, and the coding region of the inactive MB gene and the Sat2 satellite repeat, used as negative control targets. 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. These results clearly show an enrichment of the H3K9/14 double acetylation at the promoters of active genes.

Dot Blot analysis was performed to test the cross reactivity of ab232952 against Histone H3 (acetyl K9 + K14) with peptides containing other histone modifications and the unmodified H3K9/14 sequence. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. ab232952 was used at 1/20000 dilution. ab232952 shows a high specificity for the modification of interest.

Anti-Histone H3 (acetyl K9 + K14) antibody (ab232952) at 1/1000 dilution + HeLa (human epithelial cell line from cervix adenocarcinoma) histone extract at 15 µg

Predicted band size: 15 kDa

NIH/3T3 (mouse embryo fibroblast cell line) cells stained for Histone H3 (acetyl K9 + K14) (green) using ab232931 at 1/200 dilution in ICC/IF. 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 ab232952 (left) at 1/500 dilution 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.