Measuring Protein-Ligand Binding by Thermal Shift Assay.

Measuring the affinity of protein-ligand interactions is crucial to understanding protein function and facilitating drug discovery. Here, we present two methods for determining protein-ligand binding affinities using a thermal shift assay (TSA) with purified protein and a single ligand concentration. One approach assumes zero heat capacity (ZHC) change over small temperature ranges. In the second approach, the unfolding equilibrium constant (UEC) is determined directly from the unfolding curves, reducing calculations.