To determine the titer, an ELISA was performed using a serial dilution of the antibody directed against human H3K9me3S10p (bs-53072R). 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 1:87,000.


A Dot Blot analysis was performed to test the cross-reactivity of the antibody against H3K9me3S10p (bs-53072R) with peptides containing other modifications and unmodified sequences of histone H3. 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 1:1,000. The figure shows a high specificity of the antibody for the modification of interest.


HeLa cells were treated with colcemid to block the cell cycle in metaphase and 15 μg of histone extracts of these cells were analyzed by Western blot with the antibody against H3K9me3S10p (bs-53072R) diluted 1:500 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. The result of the Western analysis with the antibody is shown in lane 2; the same analysis after incubation of the antibody with 5 nmol blocking peptide for 1 hour at room temperature is shown in lane 1.


HeLa cells were stained with H3K9me3S10p Polyclonal Antibody (bs-53072R) 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 H3K9me3S10p antibody (left) diluted 1:500 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.