Anti-p53 antibody (ab232930) at 1/2000 dilution + HeLa (human epithelial cell line from cervix adenocarcinoma) nuclear extract at 40 µg

Predicted band size: 44 kDa

ChIP assays were performed using U-2 OS cells, treated with camptothecin, the ab232930 against p53 and optimized PCR primer sets for QPCR. ChIP was performed on sheared chromatin from 4 million cells. A titration of ab232930 consisting of 1, 2, 5, and 10 μg per ChIP experiment was analysed. IgG (2 μg/IP) was used as negative IP control. QPCR was performed with primers for the p21 and GAS6 genes used as positive controls, and for GAPDH gene and the Sat2 satellite repeat, used as negative controls. 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 on sheared chromatin from 4 million U-2 OS (human bone osteosarcoma epithelial cell line) cells using 1 μg of ab232930 against p53. The IP’d DNA was subsequently analysed on an Illumina HiSeq. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 51 bp tags were aligned to the human genome using the BWA algorithm. The figure shows the peak distribution along the X-chromosome (A) and in 3 genomic regions of chromosome 6, 13 and 12, surrounding p21 (CDKN1A), GAS6 and MDM2, 3 known targets genes of p53 (B, C and D, respectively).