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 B shows the peak distribution along the complete X-chromosome and in two regions surrounding the ACTB and EIF4A2 positive control genes, respectively (figure C and D).

ChIP results obtained with ab195417 against Histone H3 (acetyl K27).

ChIP assays were performed using HeLa cells, ab195417 against Histone H3 (acetyl K27) and optimized PCR primer sets for qPCR. ChIP was performed 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 ACTB and EIF4A2 genes, used as positive controls, and for the coding region of the inactive MYT1 and TSH2B genes, used as negative controls. Figure 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 Histone H3 (acetyl K27) acetylation is enriched at the promoters of active genes.

Immunofluorescent analysis of HeLa cells labeling Histone H3 (acetyl K27) with ab195417 at 1/500 diution. Cells were fixed with 4% formaldehyde for 10 min and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labelled with ab195417 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.

ChIP was performed on sheared chromatin from 1 million HeLaS3 cells using 2 μg of ab195417 against Histone H3 (acetyl K27). Quantitative PCR was performed with primers for the promoter of the active ACTB and EIF4A2 genes, used as positive controls, and for the coding region of the inactive MYT1 and MB genes, used as negative controls (Figure A). 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.

Dot Blot analysis was performed to test the cross reactivity of ab195417 against Histone H3 (acetyl K27) with peptides containing other histone modifications and the unmodified H3K27 sequence. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1/25000. The figure shows a high specificity of the antibody for the modification of interest.

All lanes : Anti-Histone H3 (acetyl K27) antibody - ChIP Grade (ab195417) at 1/1000 dilution

Lane 1 : HeLa whole cell extract at 40 µg
Lane 2 : HeLa histone extract at 15 µg
Lane 3 : Recombinant Histone H2A at 1 µg
Lane 4 : Recombinant Histone H2B at 1 µg
Lane 5 : Recombinant Histone H3 at 1 µg
Lane 6 : Recombinant Histone H4 at 1 µg

Predicted band size: 15 kDa

The antibody was diluted in TBS-Tween containing 5% skimmed milk.