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 pulldown activation assays.  However, these methods are time consuming, require large amount of sample, tend to not be very consistent and are not suitable for high throughput screens.  To circumvent these issues, we have developed the G-LISA™ assay (patent pending).

The G-LISA™ Advantage
The G-LISA™ assay uses a 96-well plate coated with RBD 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 (5-25 µg cell protein), yield quantitative and accurate data and are suitable for high thoughput screens (see Table 1).

Figure 1: The simple procedure of the G-LISA™ assay

Table 1: Comparison of traditional pulldown assay with G-LISA™

*: Clarification is still recommended for low sample numbers.
HTS applications that omit clarification has been developed

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.

Table 2: Comparison of chemiluminesce and absorbance based G-LISA™

*: Determined by titrating the amount of recombinant Rho added to the wells

Currently available list of G-LISA kits:

Rac1 specific Activation Assay (luminescence) Cat. # BK126.

Rac1,2,3 Activation Assay (colorimetric) Cat. # BK125.

RhoA specific Activation Assay (colorimetric) Cat. # BK124.

RhoA specific Activation Assay (luminescence) Cat. # BK121.

Cdc42 Activation Assay (colorimetric) Cat. # BK127.

BK122 G-LISA™ for Rho-family :: Effector Interaction Drug Discovery
In addition to our in vivo activation kits, we provide in vitro kits for drug discovery.  With these kits, you can screen for compounds that inhibit or promote the interaction between a Rho protein and its effectors.  See the in vitro G-LISA™ page for more information.

Uses:
1.   Simple, quick and reliable determination of the activation level of Rho-family small G-protein in cells or tissues.

2.   High throughput screens for Rho activity inhibitors.  Please inquire for significant discounts on large quantities of any of these kits.

Example Results:
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™ technical tips page).  All kits give results that are similar to those seen with pulldown 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).  Numbers above LPA bars correspond to fold activation compared to the control serum starved samples.

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 4h in serum free medium at 37°C, then activated with 100µg/ml calpeptin for 10min.  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 4h results in almost complete (90%) inhibition of RhoA activity.

Figure 4. Time curve of RhoA activation by LPA in Swiss 3T3 cells. Serum starved Swiss 3T3 cells were stimulated with LPA 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. Activity peaks at 2-3 min and quickly declines to levels that are just slightly above basal activity thereafter.

G-LISA™ Products:
Rac1 G-LISA™ Activation Assay, luminescence format (Cat.# BK126)
Rac1,2,3 G-LISA™ Activation Assay, colorimetric format (Cat.# BK125)
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)

Associated Products:
Anti-Cdc42 monoclonal antibody (Cat.# ACD03)
Anti-Rac1 monoclonal antibody (Cat.# ARC03)
Anti-RhoA monoclonal antibody (Cat.# ARH03)