Journal article

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



Publication Details
Authors:
Manoharan, G.; Enkvist, E.; Kasari, M.; Viht, K.; Zenn, H.; Prinz, A.; Filhol, O.; Herberg, F.; Uri, A.
Publisher:
ACADEMIC PRESS INC ELSEVIER SCIENCE

Publication year:
2015
Journal:
Analytical Biochemistry
Pages range :
10-17
Volume number:
481
Start page:
10
End page:
17
Number of pages:
8
ISSN:
0003-2697
eISSN:
1096-0309
DOI-Link der Erstveröffentlichung:


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.


Keywords
Cell lysate, FRET, Photo luminescent probes, Protein kinases, Red fluorescent protein, TR FRET

Last updated on 2024-03-12 at 10:20