Quantitative analysis of biomolecular interactions with Microscale Thermophoresis (MST)

Piotr Wardega

NanoTemper Technologies GmbH, Flößergasse 4, 81369 München, Germany


Microscale Thermophoresis (MST) allows for quantitative analysis of protein interactions in free solutions and with low sample consumption. The technique is based on thermophoresis, the directed motion of molecules in temperature gradients. Thermophoresis is highly sensitive to all types of binding-induced changes of molecular properties, be it in size, charge, hydration shell or conformation. In an all optical approach, thermophoresis is induced using an infrared laser for local heating, and molecule mobility in the temperature gradient is analyzed via fluorescence. In addition to fluorescence by labels or fusion proteins attached to one of the binding partners, intrinsic protein fluorescence can be utilized for MST thus allowing for label-free MST analysis. Its flexibility in assay design qualifies MST for biomolecular interactionanalysis in complex  experimental settings, which we herein demonstrate by addressing typically challenging types of binding events from various fields of life science. The interaction of small molecules and peptides with proteins is, despite the high molecular weight ratio, readily accessible via MST. Furthermore, MST assays are highly adaptable to fit to the diverse requirements of different biomolecules, e.g. membrane proteins to be stabilized in solution. The type of buffer and additives can be chosen freely. Measuring is even possible in complex bioliquids like celllysate and thus under close to in vivo conditions and without sample purification. Binding modes that are quantifiable via MST include dimerization, cooperativity and competition.