Cat. # WS03
Product Uses Include
- Measurement of the GTP/GDP ratio of Cdc42 in vitro.
- Quantitation of GTP-Cdc42 from tissue and tissue culture cell lysates.
- Inhibition of Cdc42 function in vitro and in vivo
Material
The Cdc42 binding domain (CBD) of the human Wiscott-Aldrich syndrome protein (WASP) has been expressed as a GST-fusion protein in E. coli. This protein binds binds specifically to GTP-bound, and not GDP-bound, Cdc42 proteins. The domain can therefore be used to specifically precipitate active, GTP-bound Rac and Cdc42 as well as to specifically block the activity of Rac and Cdc42 in vitro and in vivo.
The GST-PAK-PBD contains residues 67-150 of PAK. This region includes the highly conserved CRIB motif plus sequences required for the high affinity interaction with GTP-Rac and GTP-Cdc42.
The protein is supplied in a glutathione agarose bound format and is shipped lyophilized. The beads are colored for ease of use. The GST-tagged WASP-CBD protein can be released from the beads by incubation with 10 mM reduced glutathione.
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Figure 1. The brightly colored glutatione agarose beads in WS03 are easy to use. |
Purity
Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 12% SDS polyacrylamide gel. GST-WASP-CDB protein is >60% pure with the major contaminant being GST protein (see Figure 2).
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Figure 2: GST-WASP-CBD protein purity determination. A 20 µg sample of WS03 was separated by electrophoresis in a 12% SDS-PAGE system and stained with Coomassie Blue. The GST-WASP-CDB protein runs at approximately 34 kDa. |
Biological Activity
WASP-CBD protein specifically recognizes and binds the active, GTP-bound, form of Cdc42. It has a much lower affinity for the inactive, GDP-bound, form of Cdc42. When coupled to a colored glutathione sepharose matrix, the WASP-CBD protein beads become a convienent tool for assaying the activity of the Cdc42s. The quality control biological assay for WASP-CBD protein beads consists of a Cdc42 protein pulldown from human platelet extracts loaded with either GTPγS (Cat. # BS01) or GDP.
Examples of publications where this product was used:
Lunn, J. A. and Rozengurt, E. (2004). Hyperosmotic stress induces rapid focal adhesion kinase phosphorylation at tyrosines 397 and 577. Role of Src family kinases and Rho family GTPases. J. Biol. Chem. 279, 45266-45278.
| Product description | Cat. # | Amount | Price & Order |
| WASP-CBD protein agarose beads | WS03-A | 1 x 250 µg |
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| WS03-B | 3 x 250 µg |
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