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 H3K9me3 (Cat. No. bs-53114R). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk.


HeLa cells were stained with the Bioss antibody against H3K9me3 (bs-53114R) 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 H3K9me3 antibody (middle) diluted 1:1,000 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The left panel shows staining of the nuclei with DAPI. A merge of both stainings is shown on the right.


To determine the titer of the antibody, an ELISA was performed using a serial dilution of the antibody directed against human H3K9me3 (bs-53114R) in antigen-coated wells. 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 1:30,000.


A Dot Blot analysis was performed to test the cross-reactivity of the antibody against H3K9me3 (Cat. No. bs-53114R) with peptides containing other modifications and unmodified sequences of histone H3 and H4. 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.


Lane 1: Human SH-SY5Y cell lysates; Lane 2: Human MOLT4 cell lysates; Lane 3: Human 293T cell lysates probed with Histone H3 (Tri Methyl K9) Polyclonal Antibody, Unconjugated (bs-53114R) at 1:2000 dilution and 4°C overnight incubation. Followed by conjugated secondary antibody incubation at 1:20000 for 60 min at 37˚C.