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FRET-based screening assay using small-molecule photoluminescent probes in lysate of cells overexpressing RFP-fused protein kinases |
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| Authors: | Ganesh babu Manoharan Erki Enkvist Marje Kasari Kaido Viht Michael Zenn Anke Prinz Odile Filhol Friedrich W Herberg Asko Uri |
| Institution: | 1. Institute of Chemistry, University of Tartu, Tartu 50411, Estonia;2. Department of Biochemistry, University of Kassel, 34132 Kassel, Germany;3. Institut National de la Santé et de la Recherche Médicale, U1036, Grenoble, France; Commisariat à l''Energie Atomique, Institute of Life Sciences Research and Technologies, Biology of Cancer and Infection, Grenoble, France; Université Grenoble Alpes, Unité Mixte de Recherche, S1036, Grenoble, France |
| Abstract: | An assay was developed for the characterization of protein kinase inhibitors in lysates of mammalian cells based on the measurement of FRET between overexpressed red fluorescent protein (TagRFP)-fused protein kinases (PKs) and luminophore-labeled small-molecule inhibitors (ARC-Photo probes). Two types of the assay, one using TagRFP as the photoluminescence donor together with ARC-Photo probes containing a red fluorophore dye as acceptor, and the other using TagRFP as the acceptor fluorophore in combination with a terbium cryptate-based long-lifetime photoluminescence donor, were used for FRET-based measurements in lysates of the cells overexpressing TagRFP-fused PKs. The second variant of the assay enabled the performance of the measurements under time-resolved conditions that led to substantially higher values of the signal/background ratio and further improved the reliability of the assay. |
| Keywords: | Protein kinases Cell lysate FRET TR FRET Red fluorescent protein Photoluminescent probes |
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