H3K27me3 Polyclonal Antibody

A Dot Blot analysis was performed to test the cross-reactivity of the Bioss antibody against H3K27me3 (Cat. No. bs-53122R) with peptides containing other modifications of histone H3 and H4 and the unmodified H3K27 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 1:5,000. Figure 4 shows a high specificity of the antibody for the modification of interest. Please note that that antibody also recognizes the modification if S28 is phosphorylated.

Mouse NIH3T3 cells were stained with the Bioss antibody against H3K27me3 (Cat. No. bs-53122R) 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 H3K27me3 antibody (left) diluted 1:200 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 H3K27me3 Polyclonal Antibody (Cat. No. bs-53122R). 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 assays were performed using human HeLa cells, H3K27me3 Polyclonal Antibody (Cat. No. bs-53122R) 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 promoters of the active genes EIF4A2 and GAPDH as negative controls, and for the coding regions of the inactive genes MYT1 and TSH2B as positive controls. Figure 1 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 H3K27me3 is preferably present at inactive genes.

ChIP was performed on sheared chromatin from 1 million HeLaS3 cells using 1 \u03bcg of H3K27me3 Polyclonal Antibody (Cat. No. bs-53122R) 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. The figure shows the enrichment in genomic regions of chromosome 6, surrounding the TSH2B gene (indicated by an arrow; fig A), of chromosome 20, surrounding the MYT1 gene (fig B), and of chromosome 2 and 3 (figure C and D).