MST measures the migration of a fluorescence-labeled molecule in a temperature gradient. The migration along the temperature gradient (thermophoresis) depends on the hydrodynamic radius (size), charge, and solvation (hydration shell). Changes in one or all of these parameters result in a relative change of the fluorescence detected when a temperature gradient is applied. Hence, MST can be used to determine binding events.
Our Monolith NT.Automated instrument allows for the determination of nanomolar to millimolar binding affinities to support screening projects. In addition, we use MST as an orthogonal method of cross-validating SPR results and to support mode-of-action studies. We have experience in characterizing protein–ligand, protein–RNA, protein–DNA, and RNA–ligand interactions by MST. For characterization of protein–ligand interactions, the protein is labeled via an amine reactive fluorophore (NHS) or a cysteine reactive fluorophore (maleimide), or via binding of a fluorescent label to the his-tag of the protein. The advantage when analyzing protein–RNA, protein–DNA, and RNA–ligand interactions is the convenience of commercially available fluorescence-labeled RNA and DNA so that no additional labeling is required.