Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery

Anal Biochem. 2004 Sep 1;332(1):153-9. doi: 10.1016/j.ab.2004.04.031.


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 (Delta Tm) 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 IC(50) values from enzymatic assays. The potential pitfalls in the data analysis of thermal shift assays are also discussed.

MeSH terms

  • Amyloid Precursor Protein Secretases
  • Aspartic Acid Endopeptidases / metabolism
  • Data Interpretation, Statistical
  • Drug Evaluation, Preclinical / methods*
  • Endopeptidases
  • Fluorescent Dyes*
  • Kinetics
  • Ligands
  • Protein Denaturation
  • Proteins / antagonists & inhibitors*
  • Temperature
  • Time Factors


  • Fluorescent Dyes
  • Ligands
  • Proteins
  • Amyloid Precursor Protein Secretases
  • Endopeptidases
  • Aspartic Acid Endopeptidases