H3K27me3 (9B7) Monoclonal Antibody

Histone extracts (15 \u03bcg) from HeLa cells were analysed by Western blot using H3K27me3 (9B7) Monoclonal Antibody (bsm-53019M) diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right; the marker (in kDa) is shown on the left.

HeLa cells were stained with H3K27me3 (9B7) Monoclonal Antibody (bsm-53019M) and 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 labelled with the H3K27me3 antibody (left) diluted 1:500 in blocking solution followed by an anti-mouse secondary antibody conjugated to Alexa594. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.

To test the specificity an ELISA was performed using a serial dilution of H3K27me3 (9B7) Monoclonal Antibody (bsm-53019M). The wells were coated with peptides containing the unmodified H3K27 region as well as the mono-, di- and trimethylated H3K27 and the trimethylated H3K9. Figure shows a high specificity of the antibody for the peptide containing the modification of interest.

ChIP assays were performed using HeLa cells, H3K27me3 (9B7) Monoclonal Antibody (bsm-53019M) and optimized PCR primer sets for qPCR. ChIP was performed on sheared chromatin from 1 million cells. A titration of the antibody consisting of 1, 2, 5, and 10 \u03bcg per ChIP experiment was analysed. IgG (2 \u03bcg\/IP) was used as negative IP control. QPCR was performed with primers for the promoters of the active genes c-fos and GAPDH as negative controls, and for the coding regions of the inactive genes MYT1 and TSH2B as positive controls. 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 H3K27me3 is preferably present at inactive genes.