A Dot Blot analysis was performed to test the cross-reactivity of the Bioss antibody against H3S10p (Cat. No. bs-53135R) with peptides containing other modifications of histone H3 or the unmodified H3S10 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 1:20,000. 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 neighboring K9 is acetylated or trimethylated.


HeLa cells were treated with colcemid which blocks the cell cycle in metaphase and 15 μg of histone extracts of the cells were analyzed by Western blot using the Bioss antibody against H3S10p (Cat. No. bs-53135R) diluted 1:1,000 in TBS-Tween containing 5% skimmed milk.


To determine the titer, an ELISA was performed using a serial dilution of the Bioss antibody directed against H3S10p (Cat. No. bs-53135R) and the crude serum. 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 1:5,200.


Human osteosarcoma (U2OS) cells were stained with H3S10p Polyclonal Antibody (bs-53135R) and with DAPI. Cells were fixed with 3.7% formaldehyde in PBS for 20’ at RT, followed by a 20’ permeabilization with 0.5% Triton X-100 in PBS and blocked PBS/TX-100 containing 5% normal goat serum Figure A: cells were immunofluorescently labeled with the H3S10p antibody (left) diluted 1:2,000 in blocking buffer followed by an anti-rabbit antibody conjugated to Alexa568 or with DAPI (right). Figure B, C and D: staining of the cells with the H3S10p antibody after incubation of the antibody with 2 μM blocking peptide containing the unmodified H3S10 sequence, the phosphorylated H3S10, and the phosphorylated H3T11, respectively.