Small GTPases

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.

GTPase_cycle_copy_1

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).

 

For more information, click on the Documents tab above to see the datasheet, or contact Technical Support at tservice@cytoskeleton.com

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  1. Arf1 G-LISA Activation Assay Kit (Colorimetric Based) - 96 assays BK132
    G-LISA Arf1 Activation Assay Biochem Kit (Absorbance Based)
    Learn More
  2. Arf6 G-LISA Activation Assay Kit (Colorimetric Based) - 96 assays BK133
    G-LISA Arf6 Activation Assay Biochem Kit (Absorbance Based)
    Learn More
  3. BlastR Rapid Filtration Kit BLR02
    BlastR Rapid Filtration Kit (50 filters) Learn More
  4. BlastR Rapid Lysate Prep Kit BLR01
    BlastR Rapid Lysate Prep Kit (50 filters) Learn More
  5. Cdc42 G-LISA Activation Assay (Colorimetric format) - 96 assays BK127
    G-LISA Cdc42 Activation Assay Biochem Kit (colorimetric format)
    Learn More
  6. IgG Control Beads CIG01-beads
    IgG Control Beads (10 assays) Learn More
  7. NEW Signal-Seeker™ SUMOylation 1 Detection Kit (10 assay) BK165-S
    Signal Seeker SUMOylation 1 Detection Kit (10 assays, immunoprecipitation format) Learn More
  8. NEW Signal-Seeker™ SUMOylation 1 Detection Kit (30 assay) BK165
    Signal Seeker SUMOylation 1 Detection Kit (30 assays, immunoprecipitation format) Learn More
  9. Rac1,2,3 G-LISA Activation Assay (Colorimetric format) 96 assays BK125
    G-LISA Rac 1,2,3 Activation Assay Biochem Kit (colorimetric format)
    Learn More
  10. Ras G-LISA Activation Assay Kit (Colorimetric Based) - 96 assays BK131
    G-LISA Ras Activation Assay Biochem Kit (Absorbance Based) Learn More
  11. RhoA G-LISA Activation Assay Kit (Colorimetric format) 96 assays BK124
    G-LISA RhoA Activation Assay Biochem Kit (colorimetric format)
    Learn More
  12. Signal-Seeker™ Acetyl-Lysine Detection Kit (10 assay) BK163-S
    Signal Seeker Acetylation Detection Kit (10 assays, immunoprecipitation format) Learn More
  13. Signal-Seeker™ Acetyl-Lysine Detection Kit (30 assay) BK163
    Signal Seeker Acetylation Detection Kit (30 assays, immunoprecipitation format) Learn More
  14. Signal-Seeker™ Phosphotyrosine Detection Kit (10 assay) BK160-S
    Signal Seeker Phosphotyrosine Detection Kit (10 assays, immunoprecipitation format) Learn More
  15. Signal-Seeker™ Phosphotyrosine Detection Kit (30 assay) BK160
    Signal Seeker Phosphotyrosine Detection Kit (30 assays, immunoprecipitation format)
    Learn More
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