Journal
ANALYTICAL BIOCHEMISTRY
Volume 332, Issue 1, Pages 153-159Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2004.04.031
Keywords
thermal shift assay; ligand-binding affinity; BACEI; ITC
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The fluorescence-based thermal shift assay is a general method for identification of inhibitors of target proteins from compound libraries. Using an environmentally sensitive fluorescent dye to monitor protein thermal unfolding, the ligand-binding affinity can be assessed from the shift of the unfolding temperature (DeltaT(m)) obtained in the presence of ligands relative to that obtained in the absence of ligands. In this article, we report that the thermal shift assay can be conducted in an inexpensive, commercially available device for temperature control and fluorescence detection. The binding affinities obtained from thermal shift assays are compared with the binding affinities measured by isothermal titration calorimetry and with the IC50 values from enzymatic assays. The potential pitfalls in the data analysis of thermal shift assays are also discussed. (C) 2004 Elsevier Inc. All rights reserved.
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