Cdc42 protein: His tagged: human wild type

Cdc42 protein: His tagged: human wild type
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Product Uses Include

  • Cdc42 biochemistry
  • Cdc42 GTPase assays
  • Cdc42 nucleotide exchange assays
  • Cdc42 binding studies

Material
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
Purity is determined by scanning densitometry of proteins on SDS-PAGE gels. His-Cdc42 samples are >90% pure.

cd01gel

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.

Biological Activity
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.

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

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 tservice@cytoskeleton.com 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 tservice@cytoskeleton.com