High-Throughput Assay for RhoGEFs Based on the Transcreener® GDP Assay
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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 wordsFluorescence polarization GEF assay GEF inhibitor High-throughput Multiwell Homogeneous