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.
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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.
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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.
For product Datasheets and MSDSs please click on the PDF links below. For additional information, click on the FAQs tab above or contact our Technical Support department at tservice@cytoskeleton.com
Product Description
MSDS
Datasheet
Cat #
RhoA protein: His tagged: human constitutively active
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R6301
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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.
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.
Coming soon! If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com
