Western blot was performed on whole cell (25 μg, lane 1) and histone extracts (15 μg, lane 2) from HeLa cells, and on 1 μg of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the Bioss antibody against H3K27me3 (Cat. No. bs-53122R) diluted 1:500 in TBS-Tween containing 5% skimmed milk.


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’ 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 was performed on sheared chromatin from 1 million HeLaS3 cells using 1 μg of H3K27me3 Polyclonal Antibody (Cat. No. bs-53122R) as described above. The IP’d DNA was subsequently analyzed on an Illumina HiSeq. Library preparation, cluster generation, and sequencing were performed according to the manufacturer’s 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).