Rac1 activation assay with magnetic bead pulldown

The following protocol was followed: cells were cultured and rinsed twice with ice-cold PBS. Then, ice-cold leupeptin (0.5 ml per 150-mm tissue culture plate) was added, and cells were detached by scraping and lysed. Next, the cell lysates were transferred to microfuge tubes on ice, and 0.5 ml of each cell extract was aliquoted to a microfuge tube. A total of 10 µl (10 µg) of Rac1/Cdc42 Assay Reagent (PAK-1 PBD-conjugated magnetic beads) was added to each tube and incubated for 45 min at 4 °C. The beads were pelleted by centrifugation (10 s, 14,000×g, 4 °C), and the supernatant was removed and discarded. The beads were washed 3 times with leupeptin and resuspended in 40 µl of 2× Laemmli buffer. Next, 2 µl of 1 M dithiothreitol was added and boiled for 5 min, and the beads were pelleted by centrifugation. The supernatant and beads were mixed, and 20 µl of the mixture was loaded on a polyacrylamide gel for SDS-PAGE. After that, the proteins were transferred to a membrane. After the above steps, the membranes were blocked with 5% skim milk (w/v) at room temperature for 1 h and incubated overnight at 4 °C with an anti-Rac1 antibody (diluted to 1 µg/ml). Secondary antibodies were then added and incubated for 1 h at room temperature. Protein-antibody complexes were then detected by chemiluminescence (Pierce ECL Western Blotting Substrate, Thermo, USA).