As is already known, multiple families of small GTPases (Ras, Arf and Rho families) make up the Ras GTPase superfamily. Due to their role as molecular switches in cell signalling (active GTP-bound state vs inactive GDP-bound states) and in many other cellular responses (cytoskeletal reorganization, regulation of transcription, apoptosis…), these small GTPases are the subject of intense investigation to try to understand the mechanisms that regulate activation and inactivation of this proteins.

In this post, I invite you to discover a revolutionary way to study these cell signalling events by quantifying the level of active small GTPases in your experimental model.

Small GTP-binding proteins such as Rho and Ras GTPase family members are involved in regulating cell signalling pathways and impact a wide range of cellular processes, functions, and morphology. In this post, you’ll discover two types of Small G-protein Activation Assays to easily measure the GTP-bound form of your protein of interest, as well as some tips for choosing between both solutions and finding the assays that fit best with your cell signalling experiments.

In my previous post Small GTPases: Measuring small G protein activation I looked at state-of the-art methods for measuring the activation of small G proteins, such as RhoA, Rac1, Cdc42, and the proto-oncogen Ras. Today, I invite you to explore some methods for measuring the activity of Guanine nucleotide Exchange Factors (GEFs).

Ras and Rho family members are small G proteins involved in the regulation of actin-dependent cell processes such as motility, growth, and intracellular trafficking. Furthermore, dysfunctions of Ras and Rho proteins are known to be correlated with a number of diseases (cancer, neurodegeneration).

Small G proteins cycle between the inactive, GDP-bound form and the active, GTP-bound form.

G-LISA technology: state of the art small G protein activation measurement

Cytoskeleton, Inc. offers activation kits for a number of small G proteins (RhoA, Rac1, Cdc42, Ras, RalA, Arf1, Arf6). All these assays are available as G-LISA formats, a 96 well based technology, in which a protein sequence specifically binding to the activated for of the respective small G protein is coupled to the bottom of the wells and “catches” activated, GTP-bound proteins from cell lysates derived from cultured cells. The activation status of the small G protein can thus be detected in an ELISA like, quantitative approach.

A number of recently published papers using Cytoskeleton’s G-LISA kits show that not only RhoA and Rac1 can be measured with the RhoA-G-LISA and Rac1 G-LISA, respectively. By changing the antibody which is finally used to detect the activated small G protein bound to the binding protein one can e.g. differentiate between the isoforms RhoA, RhoB, and RhoC, and even RhoJ (which shows a high homology with Cdc42).

In their November newsletter Cytoskeleton Inc. summarized these publications and give valuable information and tips how to broaden the target specificity of their G-LISA kits.

Download your free copy of the newsletter GTPase Activation Assays: Detecting Different Isoforms

Any questions about using G-LISA? Fire away below!