Rac1 and Vinculin Detection in HeLa Cells

HeLa cells were transiently co-transfected with LOVTRAP plasmids for 20 hours before lysis in RIPA buffer. The cell extracts were adjusted to the same amount of total cellular protein (50 μg), and electrophoresed in a 4% to 15% gradient polyacrylamide gel. After electrophoretic transfer to a PVDF membrane at 1.0A for 30 min, the membranes were blocked with TBST (10mM Tris-HCl, pH7.5, 150mM NaCl, 0.05%Tween-20) containing 5% BSA for 1 hour at room temperature. The primary antibodies (mouse monoclonal anti-Rac1 antibody from Cytoskeleton, Inc., Cat. # ARC03, 1:500 dilution²⁸ ; mouse monoclonal anti-GFP Antibody from Clontech, Cat# 632381, 1:1000 dilution²⁹ ; and mouse monoclone anti-human vinculin antibody Clone h-VIN1 from Sigma, Cat# V9131, 1:200 dilution³⁰ ) in TBST were placed on the membrane and incubated at 4°C overnight. After three washings with TBST over 5 min, the second antibody (Anti-mouse IGG, conjugated with DyLight680, from Cell Signaling, Cat#5470P, 1:10,000 dilution^{31 (link)}) was applied. After three washings with TBST over 5 min, the membrane was scaned by the Odyssey® Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).