H3K27me3S28p Polyclonal Antibody

A Dot Blot analysis was performed to test the cross-reactivity of the Bioss antibody against H3K27me3S28p (Cat. No. bs-53132R) with peptides containing other modifications of histone H3 and H4 and the unmodified H3K27S28 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 1:2,000.

To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Bioss antibody directed against H3K27me3S28p (Cat. No. bs-53132R). 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 1:4,300.

ChIP assays was performed using HeLa cells treated with colcemid, H3K27me3S28p Polyclonal Antibody (Cat. No. bs-53132R) and optimized PCR primer sets for qPCR. ChIP was performed with a ChIP-seq kit, using sheared chromatin from 1 million cells. A titration of the antibody consisting of 1, 2, 5, and 10 \u03bcg per ChIP experiment was analyzed. IgG (2 \u03bcg\/IP) was used as negative IP control. QPCR was performed with primers for the promoter of the active gene GAPDH as a negative control and for the coding regions of the inactive genes MYT1 and TSH2B as positive controls. 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 H3K27me3S28p is preferably present at inactive genes.

ChIP was performed on sheared chromatin from 1.5 million colcemid treated HeLaS3 cells using 2 \u03bcg of the Bioss antibody against H3K27me3S28p (Cat. No. bs-53132R) as described above. 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 in genomic regions surrounding the TSH2B and MYT1 positive control genes (figure A and B, indicated by an arrow), and of chromosome 3 and 20 (figure C and D).