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Anal Biochem. 2015 Jul 15;481:10-7. doi: 10.1016/j.ab.2015.04.009. Epub 2015 Apr 10.

FRET-based screening assay using small-molecule photoluminescent probes in lysate of cells overexpressing RFP-fused protein kinases.

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Institute of Chemistry, University of Tartu, Tartu 50411, Estonia.
Department of Biochemistry, University of Kassel, 34132 Kassel, Germany.
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.
Institute of Chemistry, University of Tartu, Tartu 50411, Estonia. Electronic address:


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.


Cell lysate; FRET; Photoluminescent probes; Protein kinases; Red fluorescent protein; TR FRET

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