Cat. # BK036

Product Uses Include

• Analysis of in vivo RhoA 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 RhoA protein activation.  The assay uses the Rho binding domain (RBD) of the Rho effector protein, Rhotekin.  The RBD motif has been shown to bind specifically to the GTP-bound form of RhoA. The fact that the RBD region of Rhotekin has a high affinity for GTP-RhoA and that Rhotekin binding results in a significantly reduced intrinsic and catalytic rate of GTP hydrolysis make it an ideal tool for affinity purification of GTP-RhoA from cell lysates.  The Rhotekin-RBD protein supplied in this kit contains Rhotekin residues 7-89 and is in the form of a GST fusion protein, which allows one to "pull-down" the Rhotekin-RBD/Rho-GTP complex with brightly colored glutathione affinity beads. The assay therefore provides a simple means of quantitating RhoA activation in cells. The amount of activated RhoA is determined by a western blot using a RhoA specific antibody.

Kit contents
The kit contains sufficient materials for 30-60 assays depending assay setup and includes reagents for positive and negative controls. The following components are included:

1. GST-tagged Rhotekin-RBD protein on colored agarose beads (Cat. # RT02)
2. RhoA-specific monoclonal antibody (Cat. # ARH01)
3. His-tagged RhoA protein (Cat. # RH01)
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
11. Manual with detailed protocols and extensive troubleshooting guide
    
    

Figure 1. The brightly colored glutatione agarose beads in BK036 makes the kit easy to use.

Equipment needed

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

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

Figure 2. Results from BK036 RhoA activation assay. Activated Rac was precipitated and detected in a Western blot using kit BK036. The first lane shows a 50 ng recombinant His-tagged RhoA standard (Rec. His-RhoA). The following lanes shows the pulldown of iinactive, GDP-loaded, (RhoA-GDP PD) or active, GTPγS-loaded, Rac (RhoA-GTP PD) from equal amounts of cell lysates.

Examples of publications where this product was used
Birukova, A. A., Liu, F., Garcia, J. G. and Verin, A. D. (2004). Protein kinase A attenuates endothelial cell barrier dysfunction induced by microtubule disassembly. Am. J. Physiol. 287, L86-93.

Cetin, S., Ford, H. R., Sysko, L. R., Agarwal, C., Wang, J., Neal, M. D., Baty, C., Apodaca, G. and Hackam, D. J. (2004). Endotoxin inhibits intestinal epithelial restitution through activation of Rho-GTPase and increased focal adhesions. J. Biol. Chem. 279, 24592-24600.

Orr, A. W., Pallero, M. A., Xiong, W. C. and Murphy-Ullrich, J. E. (2004). Thrombospondin induces RhoA inactivation through FAK-dependent signaling to stimulate focal adhesion disassembly. J. Biol. Chem. 279, 48983-48992.

Pixley, F. J., Xiong, Y., Yu, R. Y., Sahai, E. A., Stanley, E. R. and Ye, B. H. (2005). BCL6 suppresses RhoA activity to alter macrophage morphology and motility. J. Cell Sci. 118, 1873-1883.

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.

Setiadi, H. and McEver, R. P. (2003). Signal-dependent distribution of cell surface P-selectin in clathrin-coated pits affects leukocyte rolling under flow. J. Cell Biol. 163, 1385-1395.

Please check out the new version of the Rho 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)