Sc 126 ac

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.

For immunoprecipitation, cells were lysed in RIPA buffer (Sigma) supplemented with Complete Mini EDTA-free Protease Inhibitor cocktail (Roche, Basel, Switzerland) and PhosStop (Roche). About 200 μg of lysates were pre-cleared for 30 min using normal mouse or rabbit IgG (Promega) and 20 μl protein A/G beads (Santa Cruz Biotechnology) prior to incubation with agarose beads conjugated to antibodies recognizing Flag- or Myc-conjugated beads (Sigma, 15 μl). Immunoblotting was performed as described previously .
About 100 ng of indicated recombinant proteins (Figures 6e and f) were incubated in phosphate-buffered saline supplemented with Complete Mini EDTA-free Protease Inhibitor cocktail (Roche), 0.05% Triton X100, 100 mm of NaCl and 0.05% bovine serum albumin for 1 h at 4°C. About 20 μl of DO1 (p53 antibody) conjugated to agarose (Santa Cruz Biotechnology, sc-126 AC) or 20 μl of LZAP custom antiserum conjugated to Protein A/G agarose (Santa Cruz Biotechnology) were added and incubated overnight at 40°C. The beads were spun down at 3000 r.p.m. for 1 min, washed four times with phosphate-buffered saline supplemented with 0.05% Triton X100 and 100 mm of NaCl, resuspended in 2 × Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) and boiled for 3 min. Immunoblotting was performed as described.^{70 (link)}