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The Rac/Cdc42 binding domain of the human p21-activated kinase 1 protein (PAK) has been overexpressed as a GST-tagged recombinant protein in a bacterial expression system and bound to colored glutathione Sepharose beads. The recombinant protein (amino acids 67-150) includes the highly conserved PBD region (also referred to as the CRIB region) and sequences required for the high-affinity interaction with GTP-Rac and GTP-Cdc42 proteins. The recombinant protein is tagged with GST (28 kDa) at its amino terminus and has an approximate molecular weight of 34 kDa. PAK-GST protein beads (500 µg protein per tube) are supplied as a pink/purple lyophilized powder. One tube of PAK02 is sufficient for approximately 25 assays.
PAK-PBD beads selectively bind the active (GTP-bound) forms of Rac and Cdc42, showing minimal affinity for their inactive (GDP-bound) forms. A standard assay (20 µl beads, 300 µg lysate) demonstrates this selectivity by comparing GTPγS-loaded bovine brain lysate to GDP-loaded lysate. After isolating activated Rac/Cdc42, western blotting with a Rac1 or a Cdc42-specific antibody is performed. GTPγS, a non-hydrolysable GTP analog, locks Rac1 and Cdc42 in their active conformation. Running GDP and GTPγS conditions in parallel validates both the specificity and performance of the beads. This assay results in a 7-15X enhancement of active Rac 1 and Cdc42 signal in the GTPγS sample.
Cat. #PAK02
