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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 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
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).
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|Elali, Ayman et al.||Liver X receptor activation enhances blood-brain barrier integrity in the ischemic brain and increases the abundance of ATP-binding cassette transporters ABCB1 and ABCC1 on brain capillary cells||Brain Pathology||2012||ISSN 1015-6305|
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|Burakov, Anton et al.||Centrosome positioning in interphase cells||Journal of Cell Biology||2003||ISSN 0021-9525|
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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.
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