Cdc42 protein: GST tagged: human dominant negative

Cdc42 protein: GST tagged: human dominant negative
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Product Uses Include

  • Cdc42 GEF binding studies
  • Inhibition of Cdc42 GEFs in vitro
  • Inhibition of Cdc42 in vivo by microinjection

Material
The dominant negative form of the Cdc42 protein (G25K isoform) contains a threonine to asparagine substitution at residue 17. The common name for this mutant is Cdc42(T17N) (or N17Cdc42). The asparagine substitution abolishes the protein's affinity for GTP and reduces its affinity for GDP. Hence, the Cdc42(T17N) is always in either a nucletiode free state or in its inactive, GDP-bound, state. Because of this, it binds strongly to Cdc42 GEFs and it blocks wild type Cdc42 from being activated by these GEFs.

Dominant negative form of human Cdc42 protein has been expressed in a bacterial system, and is available as a GST-tagged fusion protein. The recombinant protein is 50 kDa consisting of the Cdc42 protein (22 kDa) and a 28 kDa GST tag. The tag is at the amino terminal of the protein. 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).

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. Samples are >90% pure, the major contaminant running at 28 kDa is GST protein.

Figure 1: GST-Cdc42(T17N) purity determination. 10 µg of C17G01 was run on a SDS-PAGE gel and stained with Coomassie blue

Biological Activity
Activity of Cdc42(T17N) was verified by the ability of the protein to inhibit GEF activation of wild type Cdc42 in an in vitro GEF assay (Cat # BK100).

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

Ezratty, E. J., Partridge, M. A. and Gundersen, G. G. (2005). Microtubule-induced focal adhesion disassembly is mediated by dynamin and focal adhesion kinase. Nat. Cell Biol. 7, 581-590.

Gomes, E. R., Jani, S. and Gundersen, G. G. (2005). Nuclear movement regulated by Cdc42, MRCK, myosin, and actin flow establishes MTOC polarization in migrating cells. Cell 121, 451-463.

Burakov, A., Nadezhdina, E., Slepchenko, B. and Rodionov, V. (2003). Centrosome positioning in interphase cells. J. Cell Biol. 162, 963-969.

Sokac, A. M., Co, C., Taunton, J. and Bement, W. (2003). Cdc42-dependent actin polymerization during compensatory endocytosis in Xenopus eggs. Nat. Cell Biol. 5, 727-732.

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.

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Fenteany, G., Janmey, P. A. and Stossel, T. P. (2000). Signaling pathways and cell mechanics involved in wound closure by epithelial cell sheets. Curr. Biol. 10, 831-838.

Kalinec, F., Zhang, M., Urrutia, R. and Kalinec, G. (2000). Rho GTPases mediate the regulation of cochlear outer hair cell motility by acetylcholine. J. Biol. Chem. 275, 28000-28005.

 

Question 1:  After reconstituting the lyophilized protein with water, what is the composition of the buffer the dominant-negative human Cdc42 protein is in?

Answer 1: The protein should be reconstituted to 1 mg/ml by the addition of 25 ul of distilled water. The protein will then be in the following buffer: 10 mM Tris pH 7.5, 10 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, 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.

 

Question 3:  What is the mutation that the Cdc42 protein has that renders it dominant-negative?

Answer 3: This protein has a threonine to asparagine substitution at amino acid 17.

 

 

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