Cat. # R6301
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.
Examples of publications where this product was used:
Gallo, G., Yee, H. F., Jr. and Letourneau, P. C. (2002). Actin turnover is required to prevent axon retraction driven by endogenous actomyosin contractility. J. Cell Biol. 158, 1219-1228.
34
Storey, N. M., O'Bryan, J. P. and Armstrong, D. L. (2002). Rac and Rho mediate opposing hormonal regulation of the ether-a-go-go-related potassium channel. Curr. Biol. 12, 27-33.
Zhang, X. F., Schaefer, A. W., Burnette, D. T., Schoonderwoert, V. T. and Forscher, P. (2003). Rho-dependent contractile responses in the neuronal growth cone are independent of classical peripheral retrograde actin flow. Neuron 40, 931-944.
| Product description | Cat. # | Amount | Price & Order |
| Constitutively active RhoA protein, His-tagged | R6301-A | 1 x 10 µg |
|
| R6301-C | 4 x 10 µg |
|
