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Rho GTPases pp 165-175 | Cite as

High-Throughput Assay for RhoGEFs Based on the Transcreener® GDP Assay

  • Robert G. LoweryEmail author
  • Meera Kumar
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1821)

Abstract

We describe a high-throughput screening (HTS)-compatible method for detecting GTPase exchange factor (GEF) activity based on stimulation of GDP formation by Rho GTPases. The method is based on the fact that GDP dissociation is the rate-limiting step in the Rho GTPase catalytic cycle, so by accelerating its release a GEF causes an increase in the steady-state rate of GDP formation. The Transcreener® GDP GTPase Assay, a fluorescence polarization immunoassay (FPIA), is used to detect GDP formation in a homogeneous format.

Key words

Fluorescence polarization GEF assay GEF inhibitor High-throughput Multiwell Homogeneous 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.BellBrook LabsMadisonUSA

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