A1 Vertaisarvioitu alkuperäisartikkeli tieteellisessä lehdessä
Homogeneous GTP binding assay employing QRET technology
Tekijät: Rozwandowicz-Jansen A, Laurila J, Martikkala E, Frang H, Hemmilä I, Scheinin M, Hänninen P, Härmä H
Julkaisuvuosi: 2010
Journal: Journal of Biomolecular Screening
Tietokannassa oleva lehden nimi: Journal of Biomolecular Screening
Numero sarjassa: 3
Vuosikerta: 15
Numero: 3
Aloitussivu: 261
Lopetussivu: 267
Sivujen määrä: 7
ISSN: 1087-0571
DOI: https://doi.org/10.1177/1087057109358921
Verkko-osoite: http://api.elsevier.com/content/abstract/scopus_id:77949387276
Tiivistelmä
Functional cell signaling assays have become important tools for measuring ligand-induced receptor activation in cell-based biomolecular screening. Guanosine-5'-triphosphate (GTP) is a generic signaling marker responsible for the first intracellular signaling event of the G-protein-coupled receptors (GPCRs). [S]GTPγS binding assay is the classical well-established method for measuring agonist-induced G-protein activation requiring a separation of free and bound fractions prior to measurement. Here a novel, separation-free, time-resolved fluorescence GTP binding assay has been developed based on a nonfluorescence resonance energy transfer (FRET) single-label approach and quenching of a nonbound europium-labeled, nonliydrolyzable GTP analog (Eu-GTP). The quenching resonance energy transfer (QRET) method relies on the use of Eu-GTP, providing a time-resolved fluorescent detection as an alternative to the radiolabel [S]GTPγS assay. Upon activation of recombinant human a2A-adrenoceptors (α-AR) expressed in Chinese hamster ovary cells, guanosine-5'-diphosphate is released from the α-subunit of Gi-proteins, enabling the subsequent binding of Eu-GTP. Activation of α-AR with 5 different α-AR agonists was measured quantitatively using the developed QRET GTP assay and compared to [S]GTPγS and heterogeneous Eu-GTP filtration assays. Equal potencies and efficacy rank orders were observed in all 3 assays but with a lower signal-to-background ratio and increased assay variation in the QRET assay compared to the Eu-GTP filtration and the nonhomogeneous. © 2010 Society for Biomolecular Sciences.
Functional cell signaling assays have become important tools for measuring ligand-induced receptor activation in cell-based biomolecular screening. Guanosine-5'-triphosphate (GTP) is a generic signaling marker responsible for the first intracellular signaling event of the G-protein-coupled receptors (GPCRs). [S]GTPγS binding assay is the classical well-established method for measuring agonist-induced G-protein activation requiring a separation of free and bound fractions prior to measurement. Here a novel, separation-free, time-resolved fluorescence GTP binding assay has been developed based on a nonfluorescence resonance energy transfer (FRET) single-label approach and quenching of a nonbound europium-labeled, nonliydrolyzable GTP analog (Eu-GTP). The quenching resonance energy transfer (QRET) method relies on the use of Eu-GTP, providing a time-resolved fluorescent detection as an alternative to the radiolabel [S]GTPγS assay. Upon activation of recombinant human a2A-adrenoceptors (α-AR) expressed in Chinese hamster ovary cells, guanosine-5'-diphosphate is released from the α-subunit of Gi-proteins, enabling the subsequent binding of Eu-GTP. Activation of α-AR with 5 different α-AR agonists was measured quantitatively using the developed QRET GTP assay and compared to [S]GTPγS and heterogeneous Eu-GTP filtration assays. Equal potencies and efficacy rank orders were observed in all 3 assays but with a lower signal-to-background ratio and increased assay variation in the QRET assay compared to the Eu-GTP filtration and the nonhomogeneous. © 2010 Society for Biomolecular Sciences.
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