PCNA Binding Assay with Mutant Proteins

WT and PIP motif mutant of p125 proteins were resolved in two 12% SDS/PAGE. One of the gel was developed with Coomassie Blue staining and the second one was transferred to methanol-activated PVDF membrane. The blot was first washed with blocking buffer (BLOTTO: 25 mM Tris/HCl, pH 7.4, 150 mM NaCl, 5 mM KCl, 5% fat-free milk, 1% BSA, 0.05% Tween 20) for 1 h at room temperature. Then, the blot was incubated overnight at 4°C with 10 μg/ml of PCNA in BLOTTO with constant agitation. After thorough washings with BLOTTO, the membrane was incubated with the anti-PCNA antibody (diluted 1:1000, cat#SAB2108448; Sigma–Aldrich) in BLOTTO. Subsequently, the blot was developed with horseradish peroxidase–conjugated goat anti-rabbit IgG (diluted 1:10000 in PBST, cat# A6154; Sigma–Aldrich), and developed as explained earlier [17 (link)].