HeLa cells were treated with TSA (figure on left) or with colcemid (figure on right), and 15 μg of histone extracts of these cells were analyzed by Western blot using the antibody against H3S10p (bs-53069R) diluted 1:500 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the left; the marker (in kDa) is shown on the right. The result of the Western analysis with the antibody is shown in lane 1; lane 2 shows the same analysis after incubation of the antibody with 750 pmol blocking peptide for 1 hour at room temperature.


Hela asynchronous cells were stained with the antibody against H3S10p (bs-53069R) and with DAPI. Cells were fixed with formaldehyde, permeabilized with sodium citrate and Triton X100 and blocked with PBS containing 2.5% BSA. (left) Cells were immunofluorescently labeled with the H3S10p antibody (diluted 1:200 and incubated for 1 hour at room temperature) followed by goat anti-rabbit antibody conjugated to DyLight 488. (right) The nuclei were stained with DAPI, which specifically labels DNA. Phosphorylation of H3 on serine 10 occurs on condensed chromosomes during mitosis. This explains the dense staining of one of the cells (indicated by an arrow) in the figure.


A Dot Blot analysis was performed to test the cross-reactivity of the antibody against H3S10p (bs-53069R) with peptides containing other modifications of histone H3 and H4 and with peptides containing unmodified sequences from 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 1:20,000. The figure shows a high specificity of the antibody for the modification of interest. Note that the antibody does not recognize the H3S10p modification if the H3K9ac modification is present.


To determine the titer, an ELISA was performed using a serial dilution of the antibody directed against human H3S10p (bs-53069R). 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 1:35,000