Activation Assays

G-LISA^® Small G-protein Activation Assays

G-LISA^®  – A revolutionary new way of doing Small G-protein Activation Assays!

The Rho family of small GTPases consists of at least 20 members, the most extensively characterized of which are the RhoA, Rac1 and Cdc42 proteins.  In common with other small GTPases, the Rho proteins act as molecular switches that transmit cellular signals through downstream effector proteins by alternating between active GTP-bound and inactive GDP-bound states.  The Rho family mediates a wide range of cellular responses, including cytoskeletal reorganization, regulation of transcription, membrane trafficking and apoptosis.  Therefore, understanding the mechanisms that regulate activation and inactivation of the GTPases is of great biological significance and is a subject of intense investigation.

The traditional way of measuring the level of activation of a Rho protein has been to do so-called pull-down activation assays.  However, these methods are time consuming, require large amount of sample, tend to not be very consistent and are not suitable for plate reader technology.  To address these shortcomings, we have developed the G-LISA  assay (Patent# 7,763,418 B2).

G-LISA Advantages (Click here)

Activation Data (Click here)

High Throughput Screening (Click here)

Tech Notes(Click here)

Assays and Products(Click here)

 

The G-LISA Advantage

The G-LISA assay uses a 96-well plate coated with a RBD or PAK domain of Rho-family effector proteins.  The active GTP-bound form of the Rho-family protein, but not the inactive GDP-bound form, from a biological sample will bind to the plate.  Bound active Rho-family protein is then detected by incubation with a specific primary antibody followed by a secondary antibody conjugated to HRP. The signal is then developed with OPD or chemiluminescence reagents.  These assays are simple, fast (<3 hours), require only small amounts of sample (5-50 µg cell protein) and yield quantitative and accurate data (see Table 1).

G-LISA assays are not only improving current experimental design, but serving as enabling technology, to facilitate experiments that were not possible with traditional pull-downs. For instance, cell cultures in 3D matrices grown for 10 or more days can now be analyzed for GTPase activation (1). The G-LISA assays also provide an accuracy and sensitivity of detection that allows analyses of GTPase activity in preparations previously off-limits with pull-down assays (2,3).

Recent studies have directly compared the G-LISA activation assays with pull-downs and the advantages are clear:

G-LISA assays are superior due to the ability to use small amounts of protein (1,4,5), their greater sensitivity (1,3,5) and quantitative measurements (2).

The G-LISA kits contain all the reagents needed for the activation assay.  All you need are your cell or tissue samples, a platform shaker and a microtiter plate compatible luminometer or spectrophotometer.  The kit provides enough material for 96 assays.  The affinity wells can be separated from each other, so you can run anywhere from 2-96 assays per experiment and each kit can be used for numerous separate experiments.

The G-LISA kits are available in either luminometric or colorimetric detection versions.  The assays are identical except for the final detection step.  In general: the luminometric assays are more sensitive, while the colorimetric assays have a slightly lower level of variance.  See Table 2 for comparison of the two versions of the kit.

1. Keely et al., 2007. Methods Enzymol. v 426, p 27.
2. Oliver et al., 2011. Br J Cancer. v 104, p 324.
3. Tanaka et al., 2010. Biochem Biophys Res Commun. v 399, p 677.
4. Scott et al., 2007. J Invest Dermatol. v 127, p 668.
5. Moniz et al., 2008. Cell Signal. v 20, p 1762.

Activation Data

We have tested the G-LISA kits on several mammalian cell lines and with different well-known Rho activating stimuli (see list of tested cell types on our G-LISA FAQs page).  All kits give results that are similar to those seen with pull-down assays.  Luminometric and colorimetric assays give comparable results.  Typical results are shown in Figures 2, 3 and 4.
	Figure 2. Rho activation by lysophosphatidic acid (LPA) measured by G-LISA kit BK121.  Swiss 3T3 (mouse), A431 (human) and HeLa (human) cells were serum starved followed by stimulation by LPA.  25 µg of lysates were subjected to the G-LISA assay.  Data shown are relative luminescence units (RLU) over background signal (wells incubated with lysis buffer alone instead of cell lysates). Blue = Serum Starved, Yellow = Activated.
	Figure 3. Rho activity measured in Swiss 3T3 cells treated with the Cell Permeable Rho Inhibitor (CT04) using the RhoA G-LISA Activation Assay (BK124). Serum starved Swiss 3T3 fibroblasts were untreated (no CT04) or treated with 0.20, 0.50 and 2.0 µg/ml of CT04 for 4 h in serum free medium at 37°C, then activated with 100 µg/ml calpeptin for 10 min.  Cells were then lysed and RhoA activity was measured by the RhoA G-LISA Activation Assay (Cat.# BK124).  Note: At 2.0 µg/ml CT04 for 4 h results in almost complete (90%) inhibition of RhoA activity.
	Figure 4. Time course of RhoA activation by 10 µg/ml LPA, 100 µg/ml calpeptin (Cat.# CN01) and 1 µg/ml CNF (Cat.# CN03) in Swiss 3T3 cells. Serum-starved Swiss 3T3 cells were stimulated with activator for the times shown in the graph and RhoA activation was analyzed with G-LISA kit BK121. LPA stimulation leads to a rapid and transient activation of RhoA, which  peaks at 2-3 min and quickly declines to basal levels. Activation by calpeptin leads to a signal increase between 10-30 min which declines to basal after 60 min. Activation by CNF leads to signal increase after 60 min which is stable for up to 12 h.

G-LISA Assays:

Rac1 G-LISA Activation Assay, luminescence format (Cat.# BK126)
Rac1 G-LISA Activation Assay, colorimetric format (Cat.# BK128) NEW!
Rac1,2,3 G-LISA Activation Assay, colorimetric format (Cat.# BK125)
RalA G-LISA Activation Assay, colorimetric format (Cat.# BK129) NEW!
RhoA G-LISA Activation Assay, colorimetric format (Cat.# BK124)
RhoA G-LISA Activation Assay, luminescence format (Cat.# BK121)
Cdc42 G-LISA Activation Assay, colorimetric format (Cat.# BK127)

Related Products:

Click here for products related to G-LISA activation assays

G-LISA^® is a registered trademark of Cytoskeleton, Inc (CO). All rights reserved.

Cytoskeleton's Activation Assays have been cited hundreds of times over the past decade. A select few are described here, for more citations on individual products please use the "Citations" tab on each individual product page.



Question 1: Which is the best Small G-protein activation assay for my tissue?

Question 2: Are there any citations for the G-LISA series?

Question 1: Which is the best Small G-protein activation assay for my tissue? 

Answer 1: Cytoskeleton, Inc. offers a large number of G-LISA and traditional pull-down activation assays to study the biology and biochemistry of small G-proteins.  Our G-LISA activation assays provide an improved method of measuring the activity of small G-proteins utilizing a simple and quick protocol in 96-well format to provide extremely accurate results. To complement our G-LISA line of activation assays, we also offer the most efficiently designed and complete traditional pull-down activation assays available.

The choice of assays for measuring small G-protein activation levels is based on three factors:

1. The number of assays required.
2. The accuracy that is required.
3. Isotype range required.

For an interactive guide click on one of the hyperlinks.

Our G-LISA activation assays utilize a 96 well format with 12 x 8 well strips that provide the flexibility to run 2 to 96 wells (each well is a condition/treatment) at one time.  This flexibility is especially important if any concentration, dose or time course analyses will be performed.  Pull-downs are limited to the number of wells in each gel (usually 10-15) that will be run.  The G-LISA assays are also more accurate and quantitative than pull-downs with greater sensitivity while using less material per well.  The isotype specificity depends on the antibodies being used to capture and visualize the small G-proteins.  We have designed both types of activation assays to specifically target RhoA and Rac1.  Each of these assays can be easily modified to study RhoB, RhoC, Rac2 or Rac3.  For a quick “look and see” experiment with a few treatment conditions (one drug concentration and time point), pull-downs are convenient. 

Question 2: Are there any citations for the G-LISA series?

Answer 2: Yes, the G-LISA activation assays are well-cited in the scientific literature.  Please see the “citations” tab above for links to some current citations for our RhoA, Rac and Cdc42 G-LISA activation assays.