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Anal Biochem. 2015 Jan 1;468:42-9. doi: 10.1016/j.ab.2014.09.007. Epub 2014 Sep 18.

A universal homogeneous assay for high-throughput determination of binding kinetics.

Author information

1
Assay Development-High-Throughput Screening, Department of Lead Discovery Berlin, Bayer HealthCare, 13353 Berlin, Germany.
2
Protein Technologies, Global Drug Discovery, Department of Lead Discovery Berlin, Bayer HealthCare, 13353 Berlin, Germany.
3
Assay Development-High-Throughput Screening, Department of Lead Discovery Berlin, Bayer HealthCare, 13353 Berlin, Germany. Electronic address: amaury.fernandez@bayer.com.

Abstract

There is an increasing demand for assay technologies that enable accurate, cost-effective, and high-throughput measurements of drug-target association and dissociation rates. Here we introduce a universal homogeneous kinetic probe competition assay (kPCA) that meets these requirements. The time-resolved fluorescence energy transfer (TR-FRET) procedure combines the versatility of radioligand binding assays with the advantages of homogeneous nonradioactive techniques while approaching the time resolution of surface plasmon resonance (SPR) and related biosensors. We show application of kPCA for three important target classes: enzymes, protein-protein interactions, and G protein-coupled receptors (GPCRs). This method is capable of supporting early stages of drug discovery with large amounts of kinetic information.

KEYWORDS:

Binding kinetics; High throughput; Probe competition; TR–FRET

PMID:
25240173
DOI:
10.1016/j.ab.2014.09.007
[Indexed for MEDLINE]

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