Cat. # BK034

Product Uses Include

• Analysis of in vivo Cdc42 activation.
  
  

Introduction
The Rho switch operates by alternating between an active, GTP-bound state and an inactive, GDP-bound state.  Understanding the mechanisms that regulate activation / inactivation of the GTPases is of obvious biological significance and is a subject of intense investigation.  The fact that many Rho family effector proteins will specifically recognize the GTP bound form of the protein has been exploited experimentally to develop a powerful affinity purification assay that monitors Rac and Cdc42 protein activation.  The assay uses the Cdc42/Rac Interactive Binding (CRIB) region (also called the p21 Binding Domain, PBD) of the Cdc42 / Rac effector protein, p21 activated kinase 1 (PAK).  The CRIB/PBD protein motif has been shown to bind specifically to the GTP-bound form of Rac and/or Cdc42 proteins.  The fact that the PBD region of PAK has a high affinity for both GTP-Rac and GTP-Cdc42 and that PAK binding results in a significantly reduced intrinsic and catalytic rate of hydrolysis of both Rac and Cdc42 make it an ideal tool for affinity purification of GTP-Rac and GTP-Cdc42 from cell lysates.  The PAK-PBD protein supplied in this kit corresponds to residues 67-150.  This includes the highly conserved CRIB region (aa 74-88) plus sequences required for the high affinity interaction with GTP-Rac and GTP-Cdc42.  The PAK-PBD is in the form of a GST fusion protein, which allows one to "pull-down" the PAK-PBD/GTP-Cdc42 (or GTP-Rac) complex with glutathione affinity beads.  The assay therefore provides a simple means of quantitating Rac/Cdc42 activation in cells.  The amount of activated Cdc42 is determined by a Western blot using a Cdc42 specific antibody.

Kit contents
The kit contains sufficient materials for 25-50 assays depending assay setup and includes reagents for positive and negative controls. The following components are included:

2. GST-tagged PAK-PBD protein on colored agarose beads (Cat. # PAK02)
3. Cdc42 polyclonal antibody (Cat. # ACD03)
4. His-tagged Cdc42 protein (Cat. # CD01)
5. GTPγS: (non-hydrolyzable GTP analog) (Cat. # BS01)
6. GDP
7. Cell lysis Buffer
8. Wash Buffer
9. Loading Buffer
10. STOP Buffer
11. Protease inhibitor cocktail
12. Manual with detailed protocols and extensive troubleshooting guide
    
    

Figure 1. The brightly colored glutatione agarose beads in BK034 makes the kit easy to use.

Equipment needed

1. SDS-PAGE minigel system and western blotting transfer apparatus
   
   

Example results
The Cdc42 activation assay was tested by loading the Cdc42 protein in cell lysates with either GTPγS or GDP. As expected, the GTPγS-loaded Cdc42 is very efficiently precipitated while very little GDP-loaded Cdc42 is precipitated (Fig. 2)

Figure 1. Results from BK034 Cdc42 activation assay. Activated Cdc42 was precipitated and detected in a Western blot using kit BK034. The first lane shows a 50 ng recombinant His-tagged Cdc42 standard (Rec. His-Cdc42). The following lanes shows the pulldown of iinactive, GDP-loaded, (Cdc42-GDP PD) or active, GTPγS-loaded, Cdc42 (Cdc42-GTP PD) from equal amounts of cell lysates.

Examples of publications where this product was used
Liu, X. F., Ishida, H., Raziuddin, R. and Miki, T. (2004). Nucleotide exchange factor ECT2 interacts with the polarity protein complex Par6/Par3/protein kinase Cζ (PKCζ) and regulates PKCζ activity. Mol. Cell. Biol. 24, 6665-6675.

Nur-E-Kamal, A., Ahmed, I., Kamal, J., Schindler, M. and Meiners, S. (2005). Three dimensional nanofibrillar surfaces induce activation of Rac. Biochem. Biophys. Res. Commun. 331, 428-434.

Sasai, N., Nakazawa, Y., Haraguchi, T. and Sasai, Y. (2004). The neurotrophin-receptor-related protein NRH1 is essential for convergent extension movements. Nat. Cell Biol. 6, 741-748.

Slice, L. W., Chiu, T. and Rozengurt, E. (2005). Angiotensin II and epidermal growth factor induce cyclooxygenase-2 expression in intestinal epithelial cells through small GTPases using distinct signaling pathways. J. Biol. Chem. 280, 1582-1593.

Tang, D. D. and Gunst, S. J. (2004). The small GTPase Cdc42 regulates actin polymerization and tension development during contractile stimulation of smooth muscle. J. Biol. Chem. 279, 51722-51728.

Tang, D. D., Zhang, W. and Gunst, S. J. (2005). The Adapter Protein CrkII Regulates Neuronal Wiskott-Aldrich Syndrome Protein, Actin Polymerization, and Tension Development during Contractile Stimulation of Smooth Muscle. J. Biol. Chem. 280, 23380-23389.

Please check out the new version of the Cdc42 Activation Assay and associated products:

G-LISA™ Products:
Cdc42 G-LISA™ Activation Assay, colorimetric format (Cat.# BK127)
Rac1 G-LISA™ Activation Assay, luminescence format (Cat.# BK126)
Rac1,2,3 G-LISA™ Activation Assay, colorimetric format (Cat.# BK125)
RhoA G-LISA™ Activation Assay, colorimetric format (Cat.# BK124)
RhoA G-LISA™ Activation Assay, luminescence format (Cat.# BK121)

Associated Products:
Anti-Cdc42 monoclonal antibody (Cat.# ACD03)
Anti-Rac1 monoclonal antibody (Cat.# ARC03)
Anti-RhoA monoclonal antibody (Cat.# ARH03)