Immunoprecipitation of TP53 Protein

The immunoprecipitation method has been described previously.[20 (link)] Briefly, 2x10⁷ cells were harvested after 5 days in erythroid culture and lysed in 1 ml 1X RIPA buffer on ice for 30 minutes with sonication. The lysates were centrifuged at 16,000g for 10 minutes and the supernatant was collected and used in the immunoprecipitation assay. 50 μL of TP53 antibody-conjugated agarose beads (sc-126 AC, Santa Cruz) or mouse IgG−agarose beads (A0919, Sigma) were washed three times with 1X RIPA buffer followed by centrifugation at 100g for 3 minutes at 4°C and the supernatant was discarded. The lysates were then incubated with the TP53 antibody-conjugated agarose beads or mouse IgG−agarose beads for 1 hour at room temperature with rotation. After incubation, the mixture was centrifuged at 100g for 3 minutes at 4°C and the supernatant was used for Western blotting analysis. The immunoprecipitate was washed three times with 0.9% saline. After washing, the immunoprecipitated mixture was eluted with 200 μL of 0.1M glycine. All fractions at different stages were collected and used for Western blotting assay.