A Dot Blot analysis was performed to test the cross-reactivity of the Bioss antibody against H4K20me3 (Cat. No. bs-53115R) with peptides containing other histone modifications and the unmodified H4K20. 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


Human osteosarcoma (U2OS) cells were stained with the Bioss antibody against H4K20me3 (Cat. No. bs-53115R) and with DAPI. Cells were fixed with ice-cold methanol for 10’ and blocked with PBS/TX-100 containing 5% normal goat serum. Figure A: cells were immunofluorescently labeled with the H4K20me3 antibody (left) diluted 1:300 in blocking solution followed by an anti-rabbit secondary antibody conjugated to Alexa568 or with DAPI (right). Figure B: staining of the cells with the H4K20me3 antibody after incubation of the antibody with blocking peptide at 5 ng/μl.


To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Bioss antibody directed against H4K20me3 (Cat. No. bs-53115R), 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 antibody was estimated to be 1:7,400.


ChIP assays were performed using HeLa cells, the Bioss antibody against H4K20me3 (Cat. No. bs-53115R) and optimized PCR primer sets for qPCR. ChIP was performed with an Auto Histone ChIP-seq kit with sheared chromatin from 1 million cells using the SX-8G IP-Star automated system. A titration of the antibody consisting of 1, 2, 5, and 10 μg per ChIP experiment was analyzed. IgG (1 μg/IP) was used as negative IP control. QPCR was performed with primers for promoters of the active genes c-fos and GAPDH, used as negative controls, and for the Sat2 satellite repeat region used as a positive control. The figure shows the recovery expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).


ChIP was performed with 1 μg of the antibody against H4K20me3 (bs-53115R) on sheared chromatin from 1 million HeLaS3 cells using a ChIP-seq kit. The IP’d DNA was analyzed by qPCR with optimized PCR primer pairs for the promoter and coding region of the active GAPDH gene, for the coding region of the ZNF510 gene and for the Sat2 satellite repeat (figure A). 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 B shows the signal distribution along the long arm of chromosome 19 and a zoom-in to an enriched region containing several ZNF repeat genes. Figure C and D show the enrichment at ZNF12 and ZNF510 on chromosome 7 and 9, respectively. These results clearly show an enrichment of H4K20me3 at ZNF repeat genes.