期刊
ANALYTICAL BIOCHEMISTRY
卷 356, 期 2, 页码 273-281出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2006.05.006
关键词
HTRF; kinase assay; kinetics; inhibitor; mechanism
The rapidly growing interest in kinases as potential targets for therapeutic intervention has prompted the development of many kinase assay technologies. One exciting example is homogeneous time-resolved fluorescence (HTRF). An HTRF assay utilizes the signal generated by the fluorescence resonance energy transfer between donor and acceptor molecules in close proximity. Dual-wavelength detection helps to eliminate media interference, and the final signal is proportional to the extent of product formation. Thus far, the reported applications of this technology for in vitro kinase assays have mainly focused on high-throughput screening. In this report, we extend the applications of HTRF technology to the areas of enzyme and inhibitor characterization, some aspects of which were previously believed impossible. We describe the methods developed for determining the kinetic parameters of an enzyme, such as K-m and k(cat), and the procedures for inhibitor mechanistic studies including ATP competitiveness and slow-binding and dissociation kinetics. These assays can be readily applied to any kinase and are valuable in advancing a program through the early stages of drug discovery. (c) 2006 Elsevier Inc. All rights reserved.
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