Chapter 8: Protein Thermal Denaturation Measurements via a Fluorescent Dye

Protein thermal denaturation by following fluorescence of a reporter probe has developed into an important method to measure protein-ligand binding and characterize protein stability. The assay, usually termed, the thermal shift assay, the differential scanning fluorimetry, or ThermoFluor®, determines protein melting temperature in the presence of various ligands, inhibitors, excipients, buffers, salts, or osmolytes. The compound dosing curves enable determination of the protein denaturation thermodynamic parameters and the ligand binding constant at various temperatures, including physiological temperature. The ThermoFluor method, when the thermal shift assay is carried out in 384 well plates, is used in high-throughput screening of drug leads in the pharmaceutical industry. The general methodology and several recent developments of the method are presented in this review.