Aufsatz in einer Fachzeitschrift
FRET-based screening assay using small-molecule photoluminescent probes in lysate of cells overexpressing RFP-fused protein kinases
Details zur Publikation
Autor(inn)en: | Manoharan, G.; Enkvist, E.; Kasari, M.; Viht, K.; Zenn, H.; Prinz, A.; Filhol, O.; Herberg, F.; Uri, A. |
Verlag: | ACADEMIC PRESS INC ELSEVIER SCIENCE |
Publikationsjahr: | 2015 |
Zeitschrift: | Analytical Biochemistry |
Seitenbereich: | 10-17 |
Jahrgang/Band : | 481 |
Erste Seite: | 10 |
Letzte Seite: | 17 |
Seitenumfang: | 8 |
ISSN: | 0003-2697 |
eISSN: | 1096-0309 |
DOI-Link der Erstveröffentlichung: |
Zusammenfassung, 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. (C) 2015 Elsevier Inc. All rights reserved.
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. (C) 2015 Elsevier Inc. All rights reserved.
Schlagwörter
Cell lysate, FRET, Photo luminescent probes, Protein kinases, Red fluorescent protein, TR FRET
