Measure changes of mobility of the molecules in microscopic temperature gradients to determine binding affinities
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Microscale Thermophoresis (MST) is a powerful new technology to quantify biomolecular interactions in a few microliter solution. The MST method is based on thermophoresis, the directed movement of molecules in a temperature gradient, which strongly depends on a variety of molecular properties such as size, charge, hydration shell or conformation. Virtually any molecule (small molecules, DNA, RNA, proteins, peptides, sugars, lipids, ribosomes etc) can be analyzed. The thermophoresis is detected and quantified using either covalently attached or intrinsic fluorophores. For example, the thermophoresis of a protein typically differs significantly from the thermophoresis of a protein–ligand complex due to binding-induced changes in size, charge and solvation energy. For deriving binding constants, multiple capillaries with constant concentrations of protein and increasing concentration of ligand are scanned consecutively and thermophoresis is detected. The analysis software is used to plot and fit the change in thermophoresis to yield a Kd. This technology has several advantages over other standard techniques to analyze interactions, such as surface plasmon resonance (SPR) and isothermal microcalorimetry (ITC). It can measure affinities in free solution without surface immobilization with low sample consumption and within sub-nM to mM range. Experiments can be carried out with a broad range of solution conditions, including detergent mixtures and complex bioliquids.