H3K9/14ac Polyclonal Antibody

ChIP was performed with 1 \u03bcg of H3K9\/14ac Polyclonal Antibody (bs-53093R) on sheared chromatin from 1 million HeLaS3 cells using an Auto Histone ChIP-seq kit on the IP-Star automated system. IgG (2 \u03bcg\/IP) was used as a negative IP control. The IP\u2019d DNA was analyzed by qPCR with optimized PCR primer pairs for the promoters of the active GAPDH and c-fos genes, used as positive control targets, and the coding region of the inactive MB gene and the Sat2 satellite repeat, used as negative control targets (figure A). The IP\u2019d DNA was subsequently analyzed with an Illumina Genome Analyzer. Library preparation, cluster generation, and sequencing were performed according to the manufacturer\u2019s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. The figure shows the peak distribution along the complete sequence and an 800 kb region of the X-chromosome (figure B and C) and in 100 kb regions surrounding the RBM3, GAPDH and c-fos genes (figure D, E, and F). These results clearly show an enrichment of the H3K9\/14 double acetylation at the promoters of active genes.

To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Bioss antibody directed against H3K9\/14ac (Cat. No. bs-53093R), crude serum and flow through in antigen-coated wells. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution, the titer of the purified antibody was estimated to be 1:5,900.

A Dot Blot analysis was performed to test the cross-reactivity of the Bioss antibody against H3K9\/14ac (Cat. No. bs-53093R) with peptides containing other histone modifications and the unmodified H3K9\/14 sequence. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. 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 Bioss antibody directed against H3K9\/14ac (Cat. No. bs-53093R) diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right; the marker (in kDa) is shown on the left.

Mouse NIH3T3 cells were stained with the Bioss antibody against H3K9\/14ac (Cat. No. bs-53093R) and with DAPI. Cells were fixed with 4% formaldehyde for 10\u2019 and blocked with PBS\/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H3K9\/14ac antibody (left) diluted 1:500 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.