Cdc31 Protein Extraction and Analysis

Pelleted cells were washed in PBS and frozen in liquid nitrogen. Thawed pellets were resuspended in 1 ml lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1%NP-40, 1 mM DTT, 10% glycerol and 1 mg/ml each pepstatin A, aprotinin and leupeptin) and ~100 μl of glass beads were added prior to bead beating for 1 min x 5 with 2 min on ice between beatings. Samples were spun at 5000 rpm for 2 min and the supernatant was transferred to a new tube. Protein concentration was determined using a NanoDrop Spectrophotometer (Thermo), and equivalent amounts of lysate were analyzed by SDS-PAGE followed by western blotting. The following primary antibody dilutions were used: 1:1000 anti-Cdc31 [27 (link)] and 1:5000 anti-Pgk1 (Invitrogen). Alkaline phosphatase-conjugated secondary antibodies were used at 1:10000 (Promega). Western blot band intensity was analyzed with ImageJ Gel quantification tool.