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

Ganesh Babu Manoharan; Erki Enkvist; Marje Kasari; Kaido Viht; Michael Zenn; Anke Prinz; Odile Filhol; Friedrich W. Herberg; Asko Uri

doi:10.1016/j.ab.2015.04.009

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

Ganesh Babu Manoharan, Erki Enkvist, Marje Kasari, Kaido Viht, Michael Zenn, Anke Prinz, Odile Filhol, Friedrich W. Herberg, Asko Uri^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Research › peer-review

11 Citations (Scopus)

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.

Original language	English
Pages (from-to)	10-17
Number of pages	8
Journal	Analytical Biochemistry
Volume	481
DOIs	https://doi.org/10.1016/j.ab.2015.04.009
Publication status	Published - 3 Jun 2015
Externally published	Yes

Keywords

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

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10.1016/j.ab.2015.04.009

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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.",

keywords = "Cell lysate, FRET, Photoluminescent probes, Protein kinases, Red fluorescent protein, TR FRET",

author = "Manoharan, {Ganesh Babu} and Erki Enkvist and Marje Kasari and Kaido Viht and Michael Zenn and Anke Prinz and Odile Filhol and Herberg, {Friedrich W.} and Asko Uri",

year = "2015",

month = jun,

day = "3",

doi = "10.1016/j.ab.2015.04.009",

language = "English",

volume = "481",

pages = "10--17",

journal = "Analytical Biochemistry",

issn = "0003-2697",

publisher = "Academic Press Inc.",

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AU - Manoharan, Ganesh Babu

AU - Enkvist, Erki

AU - Kasari, Marje

AU - Viht, Kaido

AU - Zenn, Michael

AU - Prinz, Anke

AU - Filhol, Odile

AU - Herberg, Friedrich W.

AU - Uri, Asko

PY - 2015/6/3

Y1 - 2015/6/3

N2 - 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.

AB - 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.

KW - Cell lysate

KW - FRET

KW - Photoluminescent probes

KW - Protein kinases

KW - Red fluorescent protein

KW - TR FRET

UR - http://www.scopus.com/inward/record.url?scp=84930276261&partnerID=8YFLogxK

U2 - 10.1016/j.ab.2015.04.009

DO - 10.1016/j.ab.2015.04.009

M3 - Article

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AN - SCOPUS:84930276261

SN - 0003-2697

VL - 481

SP - 10

EP - 17

JO - Analytical Biochemistry

JF - Analytical Biochemistry

ER -

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

Abstract

Keywords

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