Mouse NIH3T3 cells were stained with Bioss antibody against H4K5,8,12,16ac (cat. No. bs-53098R) 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 H4K5,8,12,16ac 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.


To test the cross-reactivity of the Bioss antibody against H4K5,8,12,16ac (cat. No. bs-53098R), a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H4. 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. The figure shows a high specificity of the antibody for the modification of interest.


To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Bioss antibody directed against H4K5,8,12,16ac (cat. No. bs-53098R) 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 1:21,200.


ChIP was performed with 2 μg of the Bioss antibody against H4K5,8,12,16ac (cat. No. bs-53098R) on sheared chromatin from 1 million HeLa cells using a ChIP-seq kit as described above. The IP’d DNA was subsequently analyzed on an Illumina Genome Analyzer. Library preparation, cluster generation, and sequencing were performed according to the manufacturer’s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. Figure shows the signal distribution along the complete length of chromosome 5 (figure A) and a zoomin to a 600 kb region (figure B). Figure C and D show the enrichment in two genomic regions on chromosome 3 and 12, respectively, containing EIF4A2 and GAPDH positive controls. The position of the amplicon used for validating the QPCR results is shown with an arrow.