Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell LysatesFaiza Waheed 1,2, Pamela Speight 1,2, Qinghong Dan 1,2, Rafael Garcia-Mata 3, Katalin Szaszi 1,2
1Keenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael's Hospital , 2Department of Surgery, University of Toronto, 3Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill
The method presented here describes an assay to follow activation of RhoA specific GDP/GTP Exchange Factors (GEFs) in cultured cells by making use of a mutant RhoA GST fusion protein that has high affinity for activated GEFs. GEFs are precipitated from cell lysates, detected by Western blotting and quantified by densitometry.
