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Product Uses Include：

RhoA effector and GAP identification and binding studies

Activation of RhoA effectors in vitro and in vivo

Stimulation of RhoA phenotype by microinjection into cells

Material：
       The constitutively active form of human RhoA protein contains a glutamine to leucine substitution at residue 63. The common name for this mutant is RhoA(Q63L) or L63RhoA. The leucine substitution prevents endogenous and GAP-stimulated GTPase activity of RhoA, hence the protein is always in the active, GTP-bound, state. The corresponding mutation in Ras (Ras(Q61L)) behaves as an oncogenic mutation.

       RhoA(Q63L) has been expressed in a bacterial 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 and 0.1% dextran. Protein concentration is determined by the Precision Red Advanced Protein Assay Reagent (Cat. # ADV02).

       The recombinant protein is 25 kDa, consisting of the 22 kDa RhoA constitutively active 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：
       Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 12% polyacrylamide gel. His-RhoA(Q63L) protein was determined to be 90% pure.

        Figure 1: His-RhoA(Q63L) protein purity determination. A 10 μg sample of R6101 (His-RhoA(Q63L) molecular weight approx. 25 kDa) was separated by electrophoresis in a 12% SDS-PAGE system, and stained with Coomassie Blue.

Biological Activity：
       His-RhoA(Q63L) mutant protein binds GTP but its intrinsic GTPase activity has been eliminated, resulting in a constitutively active protein. The biological assay for His-RhoA(Q63L) activity consists of a pulldown assay using Rhotekin-RBD beads (Cat. # RT02). The Rhotekin protein is an effector of RhoA and will specifically bind to active GTP-RhoA. Stringent quality control ensures that > 80% of His-RhoA(Q63L) protein can be pulled down using this method.