FRET-based screening assay using small-molecule photoluminescent probes in lysate of cells overexpressing RFP-fused protein kinases
2015
Manoharan, Ganesh babu | Enkvist, Erki | Kasari, Marje | Viht, Kaido | Zenn, Michael | Prinz, Anke | Filhol, Odile | Herberg, Friedrich W. | Uri, Asko
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.
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