p53 Antibody

Figure 1. ChIP results obtained with the Diagenode antibody directed against p53
ChIP assays were performed using human U2OS cells, treated with camptothecin, the Diagenode antibody against p53 (Cat. No. C15410083) and optimized PCR primer sets for qPCR. ChIP was performed on sheared chromatin from 4 million cells. A titration of the antibody 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 promoter and the Sat2 satellite repeat, used as negative controls. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

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Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against p53
ChIP was performed on sheared chromatin from 4 million U2OS cells using 1 µg of the Diagenode antibody against p53 (Cat. No. C15410083) as described above. 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. Figure 2 shows the peak distribution along the X-chromosome (fig 2A) and in 3 genomic regions of chromosome 6, 13 and 12, surrounding p21 (CDKN1A), GAS6 and MDM2, 3 known targets genes of p53 (fig 2B, C and D, respectively).

Figure 3. Determination of the antibody titer
To determine the titer of the antibody, an ELISA was performed using a serial dilution of Diagenode antibody directed against human p53 (Cat. No. C15410083), in antigen coated wells. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the antibody was estimated to be 1:308,000.

Figure 4. Western blot analysis using the Diagenode antibody directed against p53
Nuclear extracts of HeLa cells (40 µg) were analysed by Western blot using the Diagenode antibody against p53 (Cat. No. C15410083) diluted 1:2,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right; the marker (in kDa) is shown on the left.