Cat. # BK008

Product Uses Include

• Analysis of in vivo Ras activation.
  
  

Introduction
The Ras switch operates by alternating between an active, GTP-bound state and an inactive, GDP-bound state.  Understanding the mechanisms that regulate activation / inactivation of Ras-like GTPases is of obvious biological significance and is a subject of intense investigation.  The fact that Ras 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 Ras protein activation.  The assay uses the Ras-binding domain (RBD) of the Ras effector kinase Raf1.  The Raf-RBD domain has been shown to bind specifically to the GTP-bound form of Ras proteins.  The fact that the Raf-RBD has a high affinity for GTP-Ras and that its binding results in a significantly reduced intrinsic and catalytic rate of hydrolysis of Ras make it an ideal tool for affinity purification of GTP-Ras from cell lysates.  The Raf-RBD is in the form of a GST fusion protein, which allows one to "pull-down" the Raf-RBD/GTP-Ras complex with glutathione affinity beads.  The assay therefore provides a simple means of quantitating Ras activation in cells.  The amount of activated Ras is determined by a western blot using a Ras specific antibody.

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

1. GST tagged Raf1-RBD protein bound to colored glutathione agarose beads (Cat. # RF02).
2. p21 Ras monoclonal antibody
3. Cell lysis Buffer
4. Protease inhibitor cocktail
5. Wash Buffer
6. Loading Buffer
7. STOP Buffer
8. GTPγS (non-hydrolyzable GTP analog, Cat. # BS01)
9. GDP
10. Manual with detailed protocols and extensive troubleshooting guide.
    
    

Figure 1. The brightly colored glutatione agarose beads in kit BK008 makes the kit easy to use.