Determination of Biomolecular Interactions using Microscale Thermophoresis

Abstract

The analysis of biomolecular interactions is important in the identification of therapeutic targets and development of diagnostics, as well as providing insights into cellular processes. MicroScale Thermophoresis (MST), an immobilization-free technology, is used to quantitate biomolecular interactions (pM-mM), ranging from protein-protein interactions to small molecule-target binding. MST, the directed movement of molecules in optically generated microscopic temperature gradients, is monitored by fluorescence. This thermophoretic movement is affected by the entropy of the hydration shell around molecules and is highly sensitive to binding reactions, which affect the size, charge, conformation, and/or hydration shell. We show how MST can be used to identify and quantify interactions between biomolecules of interest: proteins, nucleic acids, ions, etc. We also demonstrate how interactions with proteins can be analyzed in a Label-Free manner using tryptophan fluorescence. With MST, one can also probe affinities in close-to-native conditions: in detergent, liposomes, cell lysate, or blood serum.