H3K79me1 Polyclonal Antibody

A Dot Blot analysis was performed to test the cross-reactivity of the Bioss antibody against H3K79me1 (Cat. No. bs-53126R) with peptides containing other histone modifications and the unmodified H3K79. 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.

To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Bioss antibody directed against H3K79me1 (Cat. No. bs-53126R). 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 1:11,500.

ChIP assays were performed using human HeLa cells, the Bioss antibody against H3K79me1 (Cat. No. bs-53126R) and optimized PCR primer pairs for qPCR. ChIP was performed using sheared chromatin from 1 million cells. A titration consisting of 1, 2, 5 and 10 \u03bcg of antibody per ChIP experiment was analyzed. IgG (2 \u03bcg\/IP) was used as a negative IP control. Quantitative PCR was performed with primers specific for a region 1 kb upstream of the promoter and the coding region of the active GAPDH gene, used as positive controls, and for exon 2 of the inactive myoglobin (MB) gene and the Sat2 satellite repeat, used as negative controls. The 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 with 1 \u03bcg of the Bioss antibody against H3K79me1 (Cat. No. bs-53126R) and the IP\u2019d DNA was subsequently analyzed on 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 a 1 Mb region of the X-chromosome (figure A and B), in 100 kb regions surrounding the GAPDH positive control and EIF4A2 genes (figure C and D).