ChIP was performed with 1 μg of ab232908 against H2A.Z (acetyl K4 + K7 + K11) 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. 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 600 kb region of the X-chromosome and in 100 kb regions surrounding the EIF4A2, ACTB and GAPDH genes. These results clearly show an enrichment of the H2A.Z acetylation at the promoters of active genes.

ChIP assays were performed using HeLa S3 cells, ab232908 against H2A.Z (acetyl K4 + K7 + K11) and optimized primer pairs for qPCR. ChIP was performed on sheared chromatin from 1 million HeLaS3 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 using primers specific for the promoters of the ACTB and EIF4A2 genes, used as positive control targets and for the coding region of the MYT1 gene, used 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 H2A.Z is present at active promoters.

ChIP was performed with 1 μg of ab232908 against H2A.Z (acetyl K4 + K7 + K11) 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. 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 shows the peak distribution along the complete sequence. These results clearly show an enrichment of the H2A.Z acetylation at the promoters of active genes.

Dot Blot analysis was performed to test the cross reactivity of ab232908 against H2A.Z (acetyl K4 + K7 + K11) with peptides containing other histone acetylations and the unmodified H2A.Z sequence. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at 1/20,000 dilution. ab232908 shows a high specificity for the modification of interest.

HeLa (human epithelial cell line from cervix adenocarcinoma) cells stained for H2A.Z (acetyl K4 + K7 + K11) (green) using ab232908 at 1/500 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 ab232908 (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.