A homogeneous method to measure nucleotide exchange by α-subunits of heterotrimeric G-proteins using fluorescence polarization

Robin E. Muller, Klara R. Klein, Stephanie Q. Hutsell, David P. Siderovski, Adam J. Kimple

Research output: Contribution to journalArticle

Abstract

The mainstay of assessing guanosine diphosphate release by the α-subunit of a heterotrimeric G-protein is the [35S]guanosine 5′-O-(3-thiotriphosphate) (GTPγS) radionucleotide-binding assay. This assay requires separation of protein-bound GTPγS from free GTPγS at multiple time points followed by quantification via liquid scintillation. The arduous nature of this assay makes it difficult to quickly characterize multiple mutants, determine the effects of individual variables (e.g., temperature and Mg2+ concentration) on nucleotide exchange, or screen for small molecule modulators of Gα nucleotide binding/cycling properties. Here, we describe a robust, homogeneous, fluorescence polarization assay using a red-shifted fluorescent GTPγS probe that can rapidly determine the rate of GTPγS binding by Gα subunits.

Original languageEnglish
Pages (from-to)621-624
Number of pages4
JournalAssay and Drug Development Technologies
Volume8
Issue number5
DOIs
StatePublished - 1 Oct 2010

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