TY - JOUR
T1 - A homogeneous method to measure nucleotide exchange by α-subunits of heterotrimeric G-proteins using fluorescence polarization
AU - Muller, Robin E.
AU - Klein, Klara R.
AU - Hutsell, Stephanie Q.
AU - Siderovski, David P.
AU - Kimple, Adam J.
PY - 2010/10/1
Y1 - 2010/10/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=77958591671&partnerID=8YFLogxK
U2 - 10.1089/adt.2010.0286
DO - 10.1089/adt.2010.0286
M3 - Article
C2 - 20662737
AN - SCOPUS:77958591671
SN - 1540-658X
VL - 8
SP - 621
EP - 624
JO - Assay and Drug Development Technologies
JF - Assay and Drug Development Technologies
IS - 5
ER -