Product Uses Include
The human RhoA protein has been produced in a bacterial expression system. The protein is supplied as a lyophilized powder. When it is reconstituted in distilled water to 1 mg/ml, the protein is in the following buffer: 2 mM Tris pH 7.6, 0.5 mM MgCl2, 0.5% sucrose, 0.1% dextran. Protein concentration is determined by the Precision Red Advanced Protein Assay Reagent, Cat. # ADV02.
The recombinant protein is approximately 27 kDa, consisting of the RhoA protein plus a histidine tag in the amino-terminus.
For other forms of RhoA as well as many other purified small G-proteins, see our main small G-protein product page.
Purity is determined by scanning densitometry of proteins on SDS-PAGE gels. His-RhoA samples are >80% pure.
Figure 1: His-RhoA protein purity determination. A 10 µg sample of RH01 (His-RhoA molecular weight approx. 27 kDa) was separated by electrophoresis in a 12% SDS-PAGE system. The protein was stained with Coomassie Blue.
The biological activity of His-RhoA is determined from its ability to catalyze the exchange of GDP for GTP. The human Dbs (DH/PH) protein (Cat. # GE01) is an exchange factor for RhoA and Cdc42, and is used with the RhoGEF exchange assay Biochem Kit™ (Cat. # BK100) to monitor the exchange ability of His-RhoA. Stringent quality control ensures that the exchange rate (Vmax) of RhoA is enhanced eight fold in the presence of an equimolar amount of hDbs.
Lee, J. H., Katakai, T., Hara, T., Gonda, H., Sugai, M. and Shimizu, A. (2004). Roles of p-ERM and Rho-ROCK signaling in lymphocyte polarity and uropod formation. J. Cell Biol. 167, 327-337.
Kulkarni, S., Goll, D. E. and Fox, J. E. (2002). Calpain cleaves RhoA generating a dominant-negative form that inhibits integrin-induced actin filament assembly and cell spreading. J. Biol. Chem. 277, 24435-24441.