H2AK5ac Polyclonal Antibody

To test the cross reactivity of H2AK5ac Polyclonal Antibody (bs-53146R), a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H2AK5. 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. Figure shows a high specificity of the antibody for the modification of interest.

HeLa cells were stained with tH2AK5ac Polyclonal Antibody (bs-53146R) 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 H2AK5ac 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.

To determine the titer of the antibody, an ELISA was performed using a serial dilution of H2AK5ac Polyclonal Antibody (bs-53146R) 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 antibody was estimated to be 1:25,000.

ChIP assays were performed using HeLa cells, H2AK5ac Polyclonal Antibody (bs-53146R) and optimized PCR primer sets for qPCR. ChIP was performed using sheared chromatin from 1.5 million cells. A titration of the antibody consisting of 0.5, 1, 2 and, 5 \u03bcg per ChIP experiment was analyzed. IgG (1 \u03bcg\/IP) was used as negative IP control. QPCR was performed with primers for a region approximately 1 kb upstream of the GAPDH and ACTB promoters, used as positive controls, and for the coding region of the inactive MYOD1 gene and the Sat2 satellite repeat, 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).

ChIP was performed on sheared chromatin from 1.5 million HeLaS3 cells using 0.5 \u03bcg of H2AK5ac Polyclonal Antibody (bs-53146R) as described previously. The IP\u2019d DNA was subsequently analyzed on an Illumina HiSeq. Library preparation, cluster generation and sequencing were performed according to the manufacturer\u2019s instructions. The 51 bp tags were aligned to the human genome using the BWA algorithm. Figure shows the enrichment along the complete sequence and a 1 Mb region of the X-chromosome (fig A and B) and in genomic regions of chromosome 7, surrounding the ACTB gene, and of chromosome 12, surrounding the GAPDH gene (fig C and D). The position of the amplicon used for ChIP-qPCR is indicated by an arrow.