美國Cytoskeleton公司自1993年成立以來，不斷擴大產(chǎn)品線，為藥物開發(fā)、信號轉導和細胞骨架方面的研究提供高質(zhì)量的產(chǎn)品。該公司專著于生產(chǎn)純化蛋白和使用方便的試劑盒，用于研究生化和細胞信號過程。G-LISATM系列試劑盒用ELISA為基礎的方法在3小時之內(nèi)快捷準確地測定小G蛋白的活性。

   

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™

	Pulldown assay	G-LISA™
Assay Time	10-12 h （2 days）	<3 h
Cell material per assay	1-2 mg protein  （100 mm plate）	1-50 µg protein  （12 to 96-well plate）
Lysate clarification needed*	Yes	No
Sample handling	Up to 10 samples	Up to 96 samples （or more）
Quantitative data	Semi	Yes
High throughput compatible	No	Yes

*: 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™

	Luminometric detection	Colorimetric detection
Assay Time	<3 h	<3 h
Cell material per assay	1-25 µg protein	2-50 µg protein
Measurement parameters	Luminometer - high gain and 100 ms read per well	Spectrophotometer 405 nm for BK123 490 nm for BK124,BK125
Detection limit*	0.25 ng small G-protein	0.50 ng small G-protein
Linear range of detection*	0.025-1.0 ng	0.05-2.0 ng
cv of 8 replicates	16%	12%
High throughput compatible	Yes	Yes

*: 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 G-LISA™ Activation Assay, colorimetric format （Cat.# BK128） NEW!

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

RalA G-LISA™ Activation Assay, colorimetric format （Cat.# BK129） NEW!

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

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

Cdc42 Activation Assay （colorimetric） Cat. # BK127.

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 （Cat. # BK122） 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.

 

 

Note: If you are planning to switch Rho activation assay formats from a pulldown assay （such as Cat. # BK036） to G-LISA™, be sure to check the antibody that you are currently using.  If you are using a RhoA specific antibody, such as Cat. # ARH03 （contained in Cat. # BK036） or an other manufacturer’s RhoA pulldown assay kit, you should choose BK121 （luminescence） or BK124 （absorbance）.