The IP’d DNA was subsequently analysed 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 zoomin 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.

ChIP results obtained with ab195479 directed against Histone H4 (tri methyl K20).

ChIP assays were performed using human HeLa cells, ab195479 and optimized PCR primer sets for qPCR. ChIP was performed with sheared chromatin from 1 million cells. A titration of the antibody consisting of 1, 2, 5, and 10 µg per ChIP experiment was analysed. 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. Figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

Immunofluorescent analysis of Human osteosarcoma (U2OS) cells labeling Histone H4 (tri methyl K20) with ab195479 at 1/300 dilution. Cells were fixed with ice cold methanol for 10 minutes and blocked with PBS/TX-100 containing 5% normal goat serum. Figure A: cells were immunofluorescently labeled with ab195479 diluted 1/300 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa568 or with DAPI (right), which specifically labels DNA. Figure B: staining of the cells with the ab195479 after incubation of the antibody with blocking peptide (concentration: 5 ng/µl).

ChIPseq results obtained with ab195479 directed against Histone H4 (tri methyl K20). 

ChIP was performed with 1 µg of ab195479 on sheared chromatin from 1 million HeLaS3 cells. The IP’d DNA was analysed 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 analysed 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.

Dot Blot analysis was performed to test the cross reactivity of ab195479 against Histone H4 (tri methyl K20) 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 1/20000. Figure shows a high specificity of the antibody for the modification of interest.

The antibody was diluted in TBS-Tween containing 5% skimmed milk.