All lanes : Anti-Histone H4 (acetyl K12) antibody (ab231678) at 1/500 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell extracts at 25 µg
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) histone extracts at 15 µg
Lane 3 : Recombinant H2A at 1 µg
Lane 4 : Recombinant H2B at 1 µg
Lane 5 : Recombinant H3 at 1 µg
Lane 6 : Recombinant H4 at 1 µg

Dilution buffer: TBS-Tween containing 5% skimmed milk.

ChIP assays were performed using HeLa (Human epithelial cell line from cervix adenocarcinoma) cells and optimized PCR primer sets for qPCR. ChIP was performed on sheared chromatin from 1,000,000 cells. A titration of the antibody consisting of 0.5, 1, 2 and 5 µg per ChIP experiment was analysed. IgG (1 µg/IP) was used as negative IP control. QPCR was performed with primers for promoter of the active GAPDH and EIF4A2 genes, used as positive controls, and for the inactive MYOD1 gene and the Sat2 satellite repeat region used as negative controls. Image shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

ChIP was performed with 0.5 µg ab231678 on sheared chromatin from 1,000,000 HeLa (Human epithelial cell line from cervix adenocarcinoma) cells. The IP’d DNA was subsequently analysed on an Illumina HiSeq 2000. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 50 bp tags were aligned to the human genome using the BWA algorithm. Image shows the peak distribution along the complete sequence and a 2 Mb region of the human X chromosome (2A and B) and in two genomic regions surrounding the GAPDH and EIF4A2 positive control genes (2C and D).

Dot Blot analysis was performed with peptides containing other histone modifcations and the unmodifed H4. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. ab231678 was used at a dilution of 1:1,000. Image shows the antibody is specific for the K12 acetylation with some slight cross reaction with K8ac.

HeLa (Human epithelial cell line from cervix adenocarcinoma) cells stained for Histone H4 (acetyl K12) using ab231678 at a dilution of 1/200 in ICC/IF (Left panel).

Cells were fxed with 4% formaldehyde for 10 minutes and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. Secondary used is an Alexa Fluor^®488-conjugated anti-Rabbit IgG. The right panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown at the bottom.