Western blot was performed on whole cell (25 μg, lane 1) and histone extracts (15 μg, lane 2) of 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 H4K5,8,12ac (cat. No. bs-53096R). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk.


To test the cross-reactivity of the Bioss antibody against H4K5,8,12ac (cat. No. bs-53096R), 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. 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 H4K5,8,12ac (cat. No. bs-53096R) 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 1:14,500.


ChIP was performed with 0.5 μg of the Bioss antibody against H4K5,8,12ac (cat. No. bs-53096R) on sheared chromatin from 100,000 K562 cells using a ChIP-seq kit. 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. The figure shows the signal distribution along the complete length of chromosome 2 (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.