Product Uses Include：

Measurement of the GTP/GDP ratio of Rac or Cdc42 in vitro.

Quantitation of GTP-Rac/Cdc42 from tissue and tissue culture cell lysates.

Material：
       The Rac/Cdc42 (p21) binding domain (PBD) of the human p21 activated kinase 1 protein (PAK) protein has been expressed as a GST-fusion protein in E. coli. This protein binds binds specifically to GTP-bound, and not GDP-bound, Rac and 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. This product is used in our Cdc42 and Rac activation assay Biochem Kits? (Cat. # BK034 and BK035, repectively). The GST-PAK-PBD is also available as a free protein (Cat. # PAK01).

       Figure 1. The brightly colored glutatione agarose beads in PAK02 are easy to use.

Purity：
       Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 12% SDS polyacrylamide gel. GST-PAK-PBD protein is >80% pure (see Figure 2).

        Figure 2: GST-PAK-PBD protein purity determination. A 10 μg sample of PAK02 was separated by electrophoresis in a 12% SDS-PAGE system and stained with Coomassie Blue. The GST-PAK-PBD protein runs at approximately 34 kDa.

Biological Activity：
       PAK-PBD protein specifically recognizes and binds the active, GTP-bound, forms of the Rac and Cdc42 proteins. It has a much lower affinity for the inactive, GDP-bound, forms of Rac and Cdc42. When coupled to a colored glutathione sepharose matrix, the PAK-PBD protein beads become a convienent tool for assaying the activity of the Rac and Cdc42 proteins. The quality control biological assay for PAK-PBD protein beads consists of a Rac protein pulldown from human platelet extracts loaded with either GTPγS (Cat. # BS01) or GDP.