H4K8ac Polyclonal Antibody

A Dot Blot analysis was performed to test the cross-reactivity of \r\nH4K8ac Polyclonal Antibody (bs-53063R) with peptides containing other modifications of histone H4 and H3 and an unmodified histone H4 sequence. One hundred to 0.2 pmol of the peptide containing the respective histone modification were spotted on a membrane. The antibody was used at a dilution of 1:20,000. The figure shows a high specificity of the antibody for the modification of interest.

Histone extracts of HeLa cells (15 \u03bcg) were analyzed by Western blot using the antibody against H4K8ac (bs-53063R) diluted 1:250 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the left; the marker (in kDa) is shown on the right. Lane 2 shows the result of the Western analysis with the antibody; lane 1 shows the same analysis after incubation of the antibody with 750 pmol blocking peptide for 1 hour at room temperature.

ChIP assays were performed using HeLa cells treated with colcemid, H4K8ac Polyclonal Antibody (bs-53063R) and optimized PCR primer sets for qPCR. ChIP was performed using sheared chromatin from 10,000 cells. A titration of the antibody consisting of 0.5, 1.5, and 5 \u03bcl per ChIP experiment was analyzed. Additionally, ChIP was performed after incubation of the antibody with 5 nmol blocking peptide for 1 hour at room temperature. IgG (5 \u03bcg\/IP) was used as negative IP control. QPCR was performed with primers for the promoter of the active genes ALDOA and MYOD. The figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).