HTS assays for small G-proteins
The activity and regulation of small G-proteins has been shown to play an important role in the development and progression of a number of diseases such as cancer, cardiovascular disorders, inflammation and neurological disorders. Targeting G-proteins, their regulators and their downstream effectors is therefore becoming an important area of drug discovery. See the main G-protein page for general information about these proteins. Cytoskeleton, Inc. provides HTS format assays to measure in vivo activities of small G-proteins as well as in vitro drug discovery HTS assays for GEFs, GAPs and G-protein - effector interactions.
Click on the assays listed below for more information.
Cytoskeleton's small G-protein products have been cited hundreds of times over the past two decades (see individual product page for a list of citations). Due to the sensitive nature of drug screening efforts not many of these publications are directly related to drug discovery. However, there are a few that are described below:
Surviladze et al., 2010. Identification of a small GTPase inhibitor using a high-throughput flow cytometry bead-based multiplex assay. J. Biomol. Screen. 15, 10-20.
Question 1: I need a particular small G-protein/GEF combination for an assay, but I don't see it in Cytoskeleton’s list of products - can you provide those?
Answer 1: Yes, in most cases, Cytoskeleton, Inc. is able to accommodate custom protein purification needs, including fulfilling requests for specific G-protein/GEF combinations. The only requirement is that an assay needs to be available for us to measure GDP/GTP exchange to confirm that the proteins are functional. For the functional assay, we use mant-GTP in a fluorophore-based GEF assay (Cat. # BK100). If the GTPase/GEF proteins are compatible with mant-GTP, then there should be no issues.
Question 2: Which is the best assay format to develop inhibitors of small G-proteins?
Answer 2: The best assay format for developing and screening G-protein inhibitors is Cytoskeleton’s G-LISA activation assays (Cat. # BK124, BK125, BK127, BK128, BK129). These assays use a 96 well plate format which allows anywhere from 2-96 reactions (wells) to be run at one time. The G-LISAs use very little material per reaction (10-50 ug protein from a 3 cm plate), take less than 3 hours and provide quantitative data. These features make G-LISAs ideal for developing inhibitors, testing different concentrations of an inhibitor or performing inhibitor time course experiments. To study GEF and GAP inhibitors or characterize potential GEFs and GAPs, we offer the RhoGEF Exchange Assay Biochem Kit (Cat. # BK100) and the RhoGAP Assay Biochem Kit (Cat. # BK105).
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