Measuring the KD of Protein–Ligand Interactions Using Microscale Thermophoresis

Microscale thermophoresis (MST) has become a widely used technique to determine the KD or EC50 of protein–ligand interactions. The method exploits the tendency of macromolecules to migrate along a thermal gradient (i.e., thermophoresis). Differences in thermophoresis as a function of the liganded state of a macromolecule can be measured and assembled into a binding curve that can be analyzed to yield KD. In this protocol, we outline a simple experiment designed for new MST users, with the goal of using readily available, inexpensive materials to plan, execute, and analyze an MST experiment.