Electrophoretic mobility shift assays

Electrophoretic mobility shift assays (EMSAs) were performed in 20 μl reaction volume containing 20 mM HEPES (pH 7.8), 0.5 mM EDTA (pH 8.0), 6 mM MgCl₂, 60 mM KCl, 0.008% Nonidet P‐40, 100 ng anti‐p53 antibody (Pab421), 1 mM DTT, 120 ng salmon sperm DNA, 1 μl glycerol, 20,000 cpm of [³²P]‐labeled double‐stranded oligonucleotide, and either 5 μl of in vitro translated protein or 5 μg of nuclear extracts from Nutlin‐treated MEFs. After 30 min incubation at room temperature, reaction mixtures were subjected to electrophoresis on a 3.5% native polyacrylamide gel (37.5:1 acrylamide/bisacrylamide) in a Tris‐borate‐EDTA buffer at 125 V for 90 min at room temperature. 10 pmoles of competitor were added to control the specificity of DNA binding. For supershift analysis, 1 μg of anti‐p53 antibody (FL393, Santa Cruz) was added. DNA–protein complexes were revealed with X‐ray films (Kodak).