期刊
ANALYTICAL BIOCHEMISTRY
卷 397, 期 2, 页码 202-207出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2009.10.028
关键词
G proteins; Eu-GTP; [S-35]GTP gamma S; High throughput; Time-resolved fluorescence; G protein-coupled receptors
资金
- Deutsche Forschungsgemeinschaft [TR-SFB11]
[S-35]GTP gamma S, 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 [S-35]GTP gamma S 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. (C) 2009 Elsevier Inc. All rights reserved.
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