Mouse monoclonal anti p53 do 1 antibody

EBV-immortalized lymphocytes from wild-type TP53 subjects or from germline TP53 mutation carriers were seeded in duplicates in 15cm dishes, at a density of 20 x 10⁶ cells/dish and treated with 0.3 µM of doxorubicin for 8h (Sigma-Aldrich). Cells were fixed by the addition of 1% formaldehyde for 10 min (Sigma-Aldrich) and then fixation was stopped by the addition of 0.125 M glycine for 5 min (Millipore). Cells were washed 3 times in PBS and then suspended in SDS lysis buffer supplemented with a protease inhibitor cocktail (EZ-ChIP kit,Millipore). Chromatin from 20 x 10⁶ cells was fragmented by sonication using a S220 ultrasonicator (Covaris) yielding genomic DNA fragments with an average size of 250 bp. Chromatin immunoprecipitation was performed with the EZ-ChIP kit (Millipore), according to the manufacturer’s instructions, using 4 µg of mouse monoclonal anti-p53 DO-1 antibody (Santa Cruz Biotechnology, Inc.). DNA was purified on Millipore columns (EZ-ChIP kit, Millipore) and then concentrated on columns from DNA clean and concentrator-10 kit (Zymo Research).

For total protein extraction, cells were pelleted and homogenized in 100 µl of RIPA buffer (25mM Tris–HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) from Pierce Biotechnology supplemented with cocktails of protease inhibitors (Sigma-Aldrich) and phosphatase inhibitors (Pierce Biotechnology). Samples were centrifuged at 11,300 g for 20 min at 4 °C to remove cellular debris and the supernatant was collected. Thirty µg of protein were resolved by 10% SDS-PAGE. Proteins were transferred to nitrocellulose membranes (Hybond-C Extra; Amersham Biosciences). The following primary antibodies were used: mouse monoclonal anti-p53 DO-1 antibody (1/2000; Santa Cruz Biotechnology, Inc.), anti-actin JLA20 antibody (1/10000; Sigma Aldrich). Membranes were incubated with peroxidase-labelled anti-mouse or anti-rabbit antibodies (1/10000) from Jackson Immunoresearch Laboratories, and signals were detected with chemiluminescence reagents using G:Box (Syngene) and GeneSnap software.