All lanes : Anti-Histone H4 (acetyl K5) antibody (ab231913) at 1/500 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell extracts at 25 µg
Lane 2 : HeLa histone extracts 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

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

ChIP assays were performed using HeLa (Human epithelial cell line from cervix adenocarcinoma) cells, ab231913 and optimized PCR primer sets for qPCR.

ChIP was performed on 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. QPCR was performed with primers for promoter of the active gene EIF4A2 and for a region 1 kb upstream of the GAPDH gene, 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).

HeLa (Human epithelial cell line from cervix adenocarcinoma) cells stained for Histone H4 (acetyl K5) using ab231913 at a dilution of 1/500 in ICC/IF.

Cells were fxed with 4% formaldehyde for 10 minutes and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofuorescently labeled with ab231913 (Top left panel) diluted in blocking solution followed by an anti- rabbit antibody conjugated to Alexa^®488. The right panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the bottom right.

To test the cross reactivity of ab231913 a 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. The antibody was used at a dilution of 1:5,000.