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.
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.
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.
