Microscale Thermophoresis (MST) to Detect the Interaction Between Purified Protein and Small Molecule

Microscale thermophoresis (MST) is a biophysical assay to quantify the interaction between molecules, such as proteins and small molecules. In recent years, the MST assay has been used to detect protein–protein and protein–drug interactions. The assay detects the interaction between molecules by quantifying the thermophoretic movement of fluorescent molecules in response to a temperature gradient. In practice, the fluorescent molecule is mixed with different concentrations of the nonfluorescent ligand, and the mixture of molecules in solution is loaded to capillaries. A temperature gradient is applied to samples in the capillaries, and the movement of the fluorescent molecule in the temperature gradient is detected and recorded. The effect of different concentrations of the nonfluorescent ligand on the movement of the fluorescent molecule is quantified to test for the interaction between molecules. If the fluorescent molecule interacts with the ligand, the molecular properties of the molecules, such as charge, size, and hydration shell, will influence the molecular motility. MST has the advantages of being quantitative and robust. In this chapter, we will use Endosidin2 and its target protein Arabidopsis thaliana EXO70A1 (AtEXO70A1), as an example to show the procedure of using MST to test the interaction between a GFP-tagged protein and a small molecule.