Product Uses Include:

Measurement of the GTP/GDP ratio of RhoA/B/C in vitro.

Quantitation of GTP-RhoA/B/C from tissue and tissue culture cell lysates.

Material:
       The Rho binding domain (RBD) of the human Rhotekin protein has been expressed as a GST-fusion protein in E. coli. This protein binds binds specifically to GTP-bound, and not GDP-bound, RhoA, RhoB and RhoC proteins. The domain can therefore be used to specifically precipitate active, GTP-bound Rho proteins as well as to specifically block the activity of these proteins in vitro and in vivo.

       The GST-Rhotekin-RBD contains residues 7-89 of Rhotekin. This region includes the sequences required for the high affinity interaction with GTP-Rho.

       The protein is supplied in a glutathione agarose bound format and is shipped lyophilized. The beads are colored for ease of use. This product is used in our RhoA pulldown activation assay Biochem Kit? (Cat. # BK036). The GST-Rhotekin-RBD is also available as a free protein (Cat. # RT01).

       Figure 1:  GST-Rhotekin-RBD protein purity determination. A 10 μg sample of PAK01 was  

Purity:
       Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 12% SDS polyacrylamide gel. GST-Rhotekin-RBD protein is ~85% pure (see Figure 2).

       Figure 2: GST-Rhotekin-RBD protein purity determination. A 20 μg sample of RT02 was separated by electrophoresis in a 12% SDS-PAGE system and stained with Coomassie Blue. The GST-Rhotekin-RBD protein runs at approximately 35 kDa. 

Biological Activity:
       The Rhotekin-RBD protein specifically recognizes and binds to the active, GTP-bound, form of Rho protein. It has a much lower affinity for the inactive, GDP-bound, form of Rho. Biological activity of Rhotekin-RBD protein is therefore determined by its selectivity for GTP-Rho protein. The assay for monitoring the GTP-bound form of Rho is a protein pulldown assay using RT02 beads and a human platelet extract loaded with GTPγS (Cat. # BS01)