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.
