Product Uses Include
The human Cdc42 protein has been produced in a bacterial expression 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, 0.1% dextran. Protein concentration is determined by the Precision Red Advanced Protein Assay Reagent, Cat. # ADV02.
The recombinant protein is 50 kDa, consisting of the 22 kDa Cdc42 protein plus a 28 kDa GST 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 is determined by scanning densitometry of proteins on SDS-PAGE gels. GST-Cdc42 samples are >90% pure.
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Figure 1: GST-Cdc42 protein purity determination. A 20 µg sample of CDG01 (GST-Cdc42 molecular weight approx. 50 kDa) was separated by electrophoresis in a 12% SDS-PAGE system. The protein was stained with Coomassie Blue.
Biological activity of the protein is determined by demonstrating that in GTP bound CDG01 associates with PAK protein while the GDP bound form of CDG01 does not associate with PAK protein in a pull down assay.
Lesser, C. F. and Miller, S. I. (2001). Expression of microbial virulence proteins in Saccharomyces cerevisiae models mammalian infection. EMBO J. 20, 1840-1849.
Slater, S. J., Seiz, J. L., Stagliano, B. A. and Stubbs, C. D. (2001). Interaction of protein kinase C isozymes with Rho GTPases. Biochemistry 40, 4437-4445.
Question 1: After reconstituting the lyophilized protein with water, what is the composition of the buffer the wild-type human Cdc42 protein is in?
Answer 1: The protein should be reconstituted to 1 mg/ml by the addition of 25 μl of distilled water per tube. The protein will then be in the following buffer: 10 mM Tris pH 7.5, 20 mM NaCl, 0.3 mM MgCl2, 1.0% sucrose, and 0.2% 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
Question 2: What is the recommended way to store the wild-type Cdc42 protein to maintain high activity?
Answer 2: 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 (e.g. 10 μg per aliquot), snap frozen in liquid nitrogen, and stored at -70°C. The protein is stable for 6 months if stored at -70°C. The protein should not be exposed to repeated freeze-thaw cycles. The lyophilized protein is stable for 1 year if stored desiccated to <10% humidity at 4°C.
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