Product Uses Include
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 as 250 µg of lyophilized product and the protein is also available in a bead bound format (Cat. # PAK02). This product is also used in our Cdc42 and Rac activation assay Biochem Kits™ (Cat. # BK034 and BK035, repectively)
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 1).
Figure 1: GST-PAK-PBD protein purity determination. A 10 µg sample of PAK01 was separated by electrophoresis in a 12% SDS-PAGE system and stained with Coomassie Blue.
The PAK-PBD protein specifically recognizes and binds to the active, GTP-bound, form of Rac and Cdc42. Biological activity of PAK-PBD protein is therefore tested by its selectivity for GTPγS loaded Rac and Cdc42 proteins
Scott, G., Leopardi, S., Printup, S. and Madden, B. C. (2002). Filopodia are conduits for melanosome transfer to keratinocytes. J. Cell Sci. 115, 1441-1451.
Question 1: Can the GST-tagged PAK protein be used to label active GTPases immunocytochemically?
Answer 1: Yes, the GST-tagged PAK-PBD (Cat. # PAK01) proteins can be used to identify activated Rac and Cdc42 small G-proteins in fixed and permeabilized cells. Others have used a GST-tagged effector protein (GST-tagged rhotekin-RBD or GST-tagged PAK-PBD) to label active Rho or Rac/Cdc42 proteins, respectively, in cells as reported in Berdeaux et al., 2004 (Active Rho is localized to podosomes induced by oncogenic Src and is required for their assembly and function. J. Cell Biol.166, 317-323), Zhao et al., 2007 (Force activates smooth muscle actin promoter activity through the Rho signaling pathway. J. Cell Sci. 120, 1801-1809), and Wozniak et al., 2005 (R-Ras controls membrane protrusion and cell migration through the spatial regulation of Rac and Rho. Mol. Biol. Cell. 16, 84-96). Briefly, cells are grown on glass coverslips, treated with control and experimental conditions, fixed, permeabilized and incubated with PAK-PBD proteins. In the case of the rhotekin protocols, simply substitute PAK for the rhotekin protein. Localization of the bound proteins is accomplished by immunocytochemical detection of the GST tag with an anti-GST antibody that will allow localization of the activated Rac or Cdc42 small G-proteins bound by PAK-PBD. Cytoskeleton has not verified this technique as a viable and accurate means of localizing activated small G-proteins and because it is not a widely utilized technique, we cannot confirm that this procedure achieves an accurate representation of activated GTPases. Please note that PAK-PBD binds to both active Rac and Cdc42 GTPases. Therefore, additional experiments such as co-localization with a total anti-Rac or Cdc42 antibody will be necessary to verify that the active protein of interest is being labeled.
Question 2: Can the GST-tagged PAK protein detect both active Rac and Cdc42 GTPases?
Answer 2: Yes, PAK is a downstream effector protein for both activated Rac and Cdc42. The same GST-PAK protein or GST-PAK beads can be used to isolate GTP-bound Rac and Cdc42 proteins. Basically when performing the activation assay based on the western blotting technique, both Rac and Cdc will be present on the blot. Probing the blot twice, once for Cdc42 and once for Rac will give you results for both in one experiment.
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