With the introduction of the G-LISA activation assays, Cytoskeleton, Inc. revolutionized how the activity of small G-proteins is measured. The advantages of G-LISA activation assays include:

Highly Sensitivity
Highly Accuracy
Simple Protocol
Results in 3 Hours
Flexible Format
Small Amount of Material Required

The Rho family of small GTPases consists of at least 20 members, the most extensively characterized of which are the Rac1, RhoA and Cdc42 proteins (1). In common with all other small GTPases, the Rho proteins act as molecular switches that transmit cellular signals through an array of effector proteins. This family is involved in a wide range of cellular responses, including cytoskeletal reorganization (2-3), regulation of transcription (4), cell migration (5), cellular transformation and metastasis (6).
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 Rho family effector proteins preferentially recognize the GTP bound form of the protein (7) has been exploited experimentally to develop a powerful affinity purification assay that monitors Rho protein activation (8).
Traditionally, this assay has been performed using a pull-down method, wherein the Rho-GTP- binding domain (RBD) of a Rho effector is coupled to agarose beads, allowing affinity based detection of the active Rho in biological samples (8). This method suffers from several drawbacks such as being time consuming, requiring large amounts of total cellular protein, being limited in the number of samples that can be handled simultaneously and yielding only semi-quantitative results.