H3K79me3 Polyclonal Antibody

Histone extracts of HeLa cells (15 \u03bcg) were analyzed by Western blot using the Bioss antibody against H3K79me3 (Cat. No. bs-53121R), diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The molecular weight marker is shown on the right; the location of the protein of interest is indicated on the left.

A Dot Blot analysis was performed to test the cross-reactivity of the Bioss antibody against H3K79me3 (Cat. No. bs-53121R) 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.The figure shows a high specificity of the antibody for the modification of interest.

To determine the titer of the antibody, an ELISA was performed using a serial dilution of Bioss antibody directed against H3K79me3 (Cat. No. bs-53121R), 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 antibody was estimated to be 1:3,500.

ChIP was performed with the Bioss antibody against H3K79me3 (Cat. No. bs-53121R) on sheared chromatin from 1 million HeLaS3 cells using an Auto Histone ChIP-seq kit and the SX-8G IP-Star automated system. A titration of the antibody consisting of 1, 2, 5 and 10 \u03bcg per ChIP experiment was analyzed. IgG (5 \u03bcg\/IP) was used as negative IP control. Quantitative PCR was performed with primers for the GAPDH promoter and for exon 2 of the inactive myoglobin gene. The figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). These results are in accordance with the observation that H3K79me3 shows a preference for active promoters.

ChIP was performed as described above using 2 \u03bcg of the Bioss antibody against H3K79me3 (bs-53121R). The IP\u2019d DNA was 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 600 kb region of the X-chromosome (figure A and B), in a 2 Mb region from chromosome 11 (figure C), and in a 100 kb region surrounding the GAPDH positive control gene (figure D).