Journal
ANALYTICAL BIOCHEMISTRY
Volume 468, Issue -, Pages 42-49Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2014.09.007
Keywords
Binding kinetics; High throughput; Probe competition; TR-FRET
Funding
- Innovative Medicines Initiative Joint Undertaking [115366]
- European Union
- European Federation of Pharmaceutical Industries and Associations (EFPIA)
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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. (C) 2014 Elsevier Inc. All rights reserved.
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