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 22 kDa, consisting of the 22 kDa Cdc42 protein plus a 6 amino acid 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 is determined by scanning densitometry of proteins on SDS-PAGE gels. His-Cdc42 samples are >90% pure.
Figure 1: His-Cdc42 protein purity determination. A 10 µg sample of CD01 (His-Cdc42 molecular weight approx. 22 kDa) was separated by electrophoresis in a 12% SDS-PAGE system. The protein was stained with Coomassie Blue.
The biological activity of CD01 is determined by its ability to exchange nucleotide. This is tested by a pulldown assay using GST-tagged PAK-1 PBD beads (Cat. # PAK02) and GTPγS (Cat. # BS01) or GDP loaded His-Cdc42. The PAK (p21 activated kinase) protein is an effector of Cdc42, and will specifically bind to the GTP bound form Cdc42. Using this assay, the amount of biologically active GTP-bound Cdc42 is determined. Stringent quality control ensures that >70% of the Cdc42 protein produced is capable of binding GTP.
Nevins, A. K. and Thurmond, D. C. (2005). A direct interaction between Cdc42 and vesicle-associated membrane protein 2 regulates SNARE-dependent insulin exocytosis. J. Biol. Chem. 280, 1944-1952.
Kulkarni, S., Goll, D. E. and Fox, J. E. (2002). Calpain cleaves RhoA generating a dominant-negative form that inhibits integrin-induced actin filament assembly and cell spreading. J. Biol. Chem. 277, 24435-24441.
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 5 mg/ml by the addition of Milli-Q water. The protein will be in the following buffer: 50 mM Tris pH 7.6, 0.5 mM MgCl2, 50 mM NaCl, 3% sucrose, and 0.6% 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: Can the wild-type human Cdc42 protein be microinjected into cells?
Answer 2: Yes, the wild-type human Cdc42 protein can be microinjected into living cells to monitor the localization of Cdc42. For microinjection protocols, please email Cytoskeleton’s Technical Support at firstname.lastname@example.org or call and talk to a scientist at 303.322.2254, ext 316.
If you have any questions concerning this product, please contact our Technical Service department at email@example.com