Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions

Methods. 2013 Mar;59(3):301-15. doi: 10.1016/j.ymeth.2012.12.005. Epub 2012 Dec 24.


Microscale thermophoresis (MST) allows for quantitative analysis of protein interactions in free solution 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, an infrared laser is used for local heating, and molecule mobility in the temperature gradient is analyzed via fluorescence. In standard MST one binding partner is fluorescently labeled. However, MST can also be performed label-free by exploiting intrinsic protein UV-fluorescence. Despite the high molecular weight ratio, the interaction of small molecules and peptides with proteins is readily accessible by MST. Furthermore, MST assays are highly adaptable to fit to the diverse requirements of different biomolecules, such as 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 cell lysate allowing close to in vivo conditions without sample purification. Binding modes that are quantifiable via MST include dimerization, cooperativity and competition. Thus, its flexibility in assay design qualifies MST for analysis of biomolecular interactions in complex experimental settings, which we herein demonstrate by addressing typically challenging types of binding events from various fields of life science.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural
  • Review

MeSH terms

  • Animals
  • Binding, Competitive
  • Dimerization
  • GRB2 Adaptor Protein / chemistry
  • Histone Methyltransferases
  • Histone-Lysine N-Methyltransferase / chemistry
  • Lasers
  • Molecular Conformation
  • Protein Binding
  • Proteins / chemistry*
  • Protozoan Proteins / chemistry
  • Rats
  • Receptor, Adenosine A2A / chemistry
  • Receptors, Neurotensin / chemistry
  • Spectrometry, Fluorescence / methods*
  • Temperature
  • Thermodynamics
  • beta-Lactamase Inhibitors
  • beta-Lactamases / chemistry


  • GRB2 Adaptor Protein
  • Proteins
  • Protozoan Proteins
  • Receptor, Adenosine A2A
  • Receptors, Neurotensin
  • beta-Lactamase Inhibitors
  • neurotensin type 1 receptor
  • Histone Methyltransferases
  • Histone-Lysine N-Methyltransferase
  • beta-Lactamases