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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.

Author information

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.
4
Institute of Chemistry, University of Tartu, Tartu 50411, Estonia. Electronic address: asko.uri@ut.ee.

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:

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

PMID:
25866074
DOI:
10.1016/j.ab.2015.04.009
[Indexed for MEDLINE]

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