VALIDATION IMAGES
A Dot Blot analysis was performed to test the cross-reactivity of the Bioss antibody against H4K8ac (cat. No. bs-53133R) with peptides containing other histone modifications and the unmodified H4K8. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000.
Histone extracts of HeLa cells (15 μg) were analyzed by Western blot using the Bioss antibody directed against H4K8ac (cat. No. bs-53133R) diluted 1:200 in TBS-Tween containing 5% skimmed milk.
To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Bioss antibody directed against H4K8ac (cat. No. bs-53133R), crude serum and flow through 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 purified antibody was estimated to be 1:16,700.
Mouse NIH3T3 cells were stained with H4K8ac Polyclonal Antibody (cat. No. bs-53133R) 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 H4K8ac 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.