Cdc42 Pulldown Activation Assay Kit

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 50 assays, depending on assay setup, and includes reagents for positive and negative controls. The following components are included:

1. GST-tagged PAK-PBD protein on colored agarose beads (Cat. # PAK02)
2. Cdc42 monoclonal antibody (Cat. # ACD03)
3. His-tagged Cdc42 protein (Cat. # CD01)
4. GTPγS: (non-hydrolyzable GTP analog) (Cat. # BS01)
5. GDP
6. Cell lysis Buffer
7. Wash Buffer
8. Loading Buffer
9. STOP Buffer
10. Protease inhibitor cocktail (Cat. # PIC02)
11. Manual with detailed protocols and extensive troubleshooting guide