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

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 accelerat...

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Bibliographic Details
Published inMethods in molecular biology (Clifton, N.J.) Vol. 1821; p. 165
Main Authors Lowery, Robert G, Kumar, Meera
Format Journal Article
LanguageEnglish
Published United States 2018
Subjects
High-throughput
Fluorescence polarization
GEF assay
Homogeneous
Multiwell
GEF inhibitor
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Summary: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.
ISSN:1940-6029
DOI:10.1007/978-1-4939-8612-5_12