Cdc42 protein: His tagged: human constitutively active

Cdc42 protein: His tagged: human constitutively active
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Product Uses Include

  • Cdc42 effector and GAP identification and binding studies
  • Activation of Cdc42 effectors in vitro and in vivo
  • Stimulation of Cdc42 phenotype by microinjection into cells

Material
The constitutively active form of human Cdc42 protein contains a glutamine to leucine substitution at residue 61. The common name for this mutant is Cdc42(Q61L) or L61Cdc42. The leucine substitution prevents endogenous and GAP-stimulated GTPase activity of Cdc42, hence the protein is always in the active, GTP-bound, state. A similar mutation in Ras (Ras(Q61L)) behaves as an oncogenic mutation.

Cdc42(Q61L) 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 Cdc42 constitutively active protein plus a histidine tag in the amino-terminus.

For other forms of Cdc42 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-Cdc42(Q61L) protein was determined to be >70% pure.

Figure 1: His-Cdc42(Q61L) protein purity determination. A 10 µg sample of C6101 (His-Cdc42(Q61L) molecular weight approx. 25 kDa) was separated by electrophoresis in a 12% SDS-PAGE system, and stained with Coomassie Blue.

Biological Activity
His-Cdc42(Q61L) mutant protein binds GTP but its intrinsic GTPase activity has been eliminated, resulting in a constitutively active protein. The biological assay for His-Cdc42(Q61L) activity consists of a pulldown assay using PAK-PBD beads (Cat. # PAK02). The PAK protein is an effector of Cdc42 and will specifically bind to active GTP-Cdc42. Stringent quality control ensures that > 80% of His-Cdc42(Q61L) 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

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.

Question 1:  Does the constitutively-active mutant Cdc42 protein come pre-loaded with GTP?

Answer 1: The purified small G-proteins that Cytoskeleton provides, whether they are wild-type, constitutively-active or dominant negative proteins, do not come pre-loaded with GTP.  Typically, 50% of the protein is bound to GTP and 50% is bound to GDP upon resuspension.  Thus, proteins will have to be pre-loaded with either GTP (Cat. # BST06) or GTPgS (Cat. # BS01) for experiments.

 

Question 2:  In what buffer is the mutant Cdc42 protein shipped?

Answer 2: The His-Cdc42 L61 mutant protein (Cat. # C6101) is supplied as a lyophilized white powder.  The protein should be reconstituted to 1 mg/ml by the addition of 10 μl of distilled water.  The protein will be in the following buffer: 10 mM Tris pH 7.5, 10 mM NaCl, 0.1 mM MgCl2, 0.5% sucrose and 0.1% dextran.  In order to maintain high biological activity of the protein, it is strongly recommended that the protein solution be supplemented with DTT to 1 mM final concentration, aliquoted into "experiment-sized" amounts, snap frozen in liquid nitrogen and stored at -70°C. The protein is stable for 6 months under these conditions. The protein must not be exposed to repeated freeze-thaw cycles.

 

If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com.