VALIDATION IMAGES
Western blot was performed on whole cell (25μg, lane 1) and histone extracts (15μg, 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 H4K5ac Polyclonal Antibody (bs-53099R). Antibody was diluted 1:500 in TBS-Tween containing 5% skim milk.
Dot Blot analysis was performed with peptides containing a variety of histone modifications and the unmodified H4. 0.2-100 pmol of the respective peptides were spotted on a membrane. H4K5ac Polyclonal Antibody (bs-53099R) was used at 1:5,000 dilution. Result shows high specificity of the antibody for the modification of interest.
ELISA was performed using a serial dilution of the
H4K5ac Polyclonal Antibody (bs-53099R) against peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:8,900.
HeLa cells were stained with the H4K5ac Polyclonal Antibody (bs-53099R) 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 H4K5ac 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 staining is shown on the right.