title: FRET-based screening assay using small-molecule photoluminescent probes in lysate of cells overexpressing RFP-fused protein kinases.
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.
authors: Institute of Chemistry, University of Tartu, Tartu 50411, Estonia.
authors: Ganesh Babu
authors: GB
authors: Manoharan
authors: Institute of Chemistry, University of Tartu, Tartu 50411, Estonia.
authors: Erki
authors: E
authors: Enkvist
authors: Institute of Chemistry, University of Tartu, Tartu 50411, Estonia.
authors: Marje
authors: M
authors: Kasari
authors: Institute of Chemistry, University of Tartu, Tartu 50411, Estonia.
authors: Kaido
authors: K
authors: Viht
authors: Department of Biochemistry, University of Kassel, 34132 Kassel, Germany.
authors: Michael
authors: M
authors: Zenn
authors: Department of Biochemistry, University of Kassel, 34132 Kassel, Germany.
authors: Anke
authors: A
authors: Prinz
authors: 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.
authors: Odile
authors: O
authors: Filhol
authors: Department of Biochemistry, University of Kassel, 34132 Kassel, Germany.
authors: Friedrich W
authors: FW
authors: Herberg
authors: Institute of Chemistry, University of Tartu, Tartu 50411, Estonia. Electronic address: asko.uri@ut.ee.
authors: Asko
authors: A
authors: Uri
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D049408:Luminescent Agents / Q000737:chemistry; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation; D000097573:Red Fluorescent Protein
chemical_list: D049408:Luminescent Agents; D008164:Luminescent Proteins; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein
keywords: Cell lysate; FRET; Photoluminescent probes; Protein kinases; Red fluorescent protein; TR FRET