Microscale Thermophoresis exploits the directed motions of biomolecules and macromolecular complexes in solution in microscopic thermal gradients.  It can be used to measure equilibrium binding events.

Thermophoretic mobility is dependent on the solvation entropy, charge, size and conformation of the molecules observed. Essentially, changes in thermophoretic mobility result from changes in the hydration shell; such changes manifest from almost any binding event, as this will change structure/conformation, size, charge, and so on.  During most binding events one or several of these properties will change, making MST a powerful technique for the quantitative analysis of binding reactions.  Thermophoretic movement is detected via a fluorescently labeled binding partner, and an infrared laser (1480 nm wavelength) is focused onto the sample spot where fluorescence intensity is measured. The focused infrared radiation creates a highly localized temperature gradient (ca. 2 – 8 degrees) in the aqueous sample solution.  In this way, thermophoresis is induced in the sample.  Typically, this results in a rapid drop in initial fluorescence followed by diffusion-limited thermophoresis that lasts several seconds.  These processes are affected by binding events.  When the IR laser is turned off, back-diffusion occurs and fluorescence recovers toward its initial level.

Using MST, you are not only able to determine affinities, but you can also assess other physical parameters such as stoichiometry, aggregation, enthalpy (van’t Hoff plot), slow enzyme kinetics  and oligomerization.

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Exciting Possibilities in Biology, Biochemistry and Biophysics:

  • Kd in the nM to mM range under near physiological condition ((protein-protein, protein-ligand, DNA/RNA-ligand, DNA/RNA-protein, protein-lipids, peptide-lipids, small molecule binding, membrane proteins in liposomes/nano discs or detergent solution ...)
  • Determine stoichiometry and number of binding sites
  • Investigate oligomerization and aggregates
  • Binding energetics ΔG (free energy), ΔH (enthalpy) and ΔS (entropy)
  • No immobilization necessary
  • Either component can be labelled and the label can be located far from the interaction site
  • No size limitations
  • Low sample consumption
  • And much more...