Product Uses Include
p50RhoGAP (sometimes called Cdc42-GAP or Rho GAP) is a GTPase Activating Protein that catalyzes the hydrolysis of GTP for Rac, Rho, and Cdc42 proteins. It appears to be most active for Cdc42 and least active for Rac.
p50RhoGAP has been purified from a bacterial expression system and is supplied as a GST-fusion protein. This protein is 78 kDa in size and is comprised of the 50 kDa full length p50RhoGAP protein and a 28 kDa GST tag. It is supplied as a lyophilized powder. When reconstituted to 1 mg/ml, the protein is in the following buffer: 2 mM Tris pH 7.6, 0.5 mM MgCl2, 0.5% sucrose and 0.1% dextran. Protein concentration is determined by the Precision Red Advanced Protein Assay Reagent cat. # ADV02.
p50RhoGAP is also available as a purified GST-tagged catalytic (GAP) domain only (Cat. # GAS01)
Purity is determined by scanning densitometry of proteins on SDS-PAGE gels. Samples are 90% pure.
Figure 1: GST-p50RhoGAP protein purity determination. A 10 µg sample of GAP01 was separated by electrophoresis in a 12% SDS-PAGE system. The protein was stained with Coomassie Blue.
Biological activity of full length p50RhoGAP were verified by the ability of the proteins to enhance GTP hydrolysis for GST-RhoA, GST-Rac1 and GST-Cdc42 proteins (Cat # RHG01, RCG01 and CDG01, respectively). At a molar ratio of 1:0.3 (G-protein : full length p50RhoGAP) hydrolysis of bound GTP was at least 75% completed after 5 min at room temperature. Hydrolysis of bound GTP in the absence of RhoGAP was 0% (GST-RhoA), 10% (GST-Cdc42) or 45% (GST-Rac1) after 5 min at room temperature. GTPase assays were performed using BK055.
Li, X., Bu, X., Lu, B., Avraham, H., Flavell, R. A. and Lim, B. (2002). The hematopoiesis-specific GTP-binding protein RhoH is GTPase deficient and modulates activities of other Rho GTPases by an inhibitory function. Mol. Cell. Biol. 22, 1158-1171.