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).

Fig. 1: Small G protein activation cycle

In this post, I invite you to discover some state-of-the art methods to measure the activation of the most prominent small G proteins. In future posts, we’ll then be looking at methods to measure the activity of Guanine nucleotide exchange factors (GEFs) which stimulate the release of GDP to allow the binding of GTP (which activates the small G protein) and of GTPase-activating proteins (GAPs) which stimulate the GTPase activity of small G proteins to inactive them (see Fig. 1).

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!

Small GTP-binding proteins such as RhoA, Rac1, and Cdc42 are involved in regulating cell signalling pathways and impact a wide range of cellular processes, functions, and morphology. They bind and hydrolyze GTP, thus being switched from the activated form to the inactivated form.

The most prominent family of small G proteins is represented by the Ras superfamily of proteins. The Rho subfamily belonging to this superfamily consists of proteins like RhoA, Rac1, and Cdd42. These proteins have been shown to be involved in the regulation of actin dynamics, thus playing a crucial role in processes like cell movement, intracellular transport, and organelle development. While RhoA affects actin stress fibers, Rac1 exhibits effects on lamellipodia and Cdc42 on filopodia.

Measuring the activation of small G proteins

In the past, the activation of small G proteins (i.e. the transformation of the GDP-bound form to the GTP-bound form) could only be measured in pull-down assays. The assays make use of effector proteins which specifically bind to the activated forms of small G proteins. Coupled to beads these proteins can be used to pull-down the activated small G protein from a cell lysate.

Cytoskeleton Inc. introduced a new format of this approach a few years ago. With the G-LISA technology, the effector proteins which selectively bind to activated small G proteins are coupled to 96 well plates, making it possible to run an ELISA-like activation assay. To help you to choose the right activation assay format for your application, take a look at this short video.

New trial sizes for the most commonly investigated small G proteins

Up to now, researchers had to order full 96 well plates to start using the G-LISA technology.

Now Cytoskeleton Inc. have launched trial kits (which are attractively priced) to enable you to establish the method in your lab:

# RhoA G-LISA Kit (Colorim.) Trial Size

# Rac1 G-LISA Activation Assay (Colorim.) Trial Size

# Cdc42 G-LISA Kit (Colorim.) Trial Size

And you can even try all 3 small G proteins with one kit!

# RhoA/Rac1/Cdc42 G-LISA Activation Assay Bundle

What about you?

Are you interested in the G-LISA technology? Or if you’re already using it, how does it compare to other methods?
Share your comments below!