Europium-labeled GTP as a general nonradioactive substitute for [(35)S]GTPgammaS in high-throughput G protein studies.
[(35)S]GTPgammaS, the nonhydrolyzable radioactive GTP analog, has been a powerful tool in G protein studies and has set the standards in this field of research. However, its radioactive nature imposes clear limitations to its use in regular laboratory practice and in high-throughput experimentation. The europium-labeled GTP analog (Eu-GTP) has been used as an alternative in the analysis of G protein activation by G protein-coupled receptors in cellular membrane preparations. Here we expand the usage of Eu-GTP and show that it can be applied in other types of assays where [(35)S]GTPgammaS has been previously utilized. We demonstrate the applicability of the modified Eu-GTP binding technology to analysis of heterotrimeric and monomeric G proteins of natural and recombinant sources, from different organisms, in assays with soluble proteins and membrane-containing assays of a high-throughput format. The deci-nanomolar K(D) of Eu-GTP for the tested G proteins is similar to that of other fluorescent-modified GTP analogs, while the sensitivity achieved in time-resolved fluorescence analysis of Eu-GTP exceeds that of the radioactive measurements. Overall, the results of our modified Eu-GTP binding assay present Eu-GTP as a general nonradioactive alternative for G protein studies, especially attractive in high-throughput experiments.
Koval
Alexey
A
University of Konstanz, Department of Biology, Universitätsstrasse 10, Box 643, Konstanz 78457, Germany.
Kopein
Damir
D
Purvanov
Vladimir
V
Katanaev
Vladimir L
VL
D000818:Animals; D005063:Europium / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D007700:Kinetics; D009942:Organometallic Compounds / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D013552:Swine
D009942:Organometallic Compounds; D043562:Receptors, G-Protein-Coupled; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D005063:Europium; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020962:Heterotrimeric GTP-Binding Proteins