Cdc42-His Protein: constituitively active

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 ug 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.

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 &mu, l 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 infohoelzel.de.