MICROSCALE THERMOPHORESIS (MST)

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Microscale thermophoresis (MST)

Microscale thermophoresis (MST) is an immobilization-free technology for quantifying molecular interactions. MST is based on thermophoresis, the directed movement of molecules in a temperature gradient. Thermophoresis strongly depends on a variety of molecular properties such as size, charge and/or solvation energy, and therefore, the thermophoresis of a protein typically differs from the thermophoresis of a protein-ligand complex.MST can be used with almost all kinds of target molecules,  in a wide range of standard buffers and complex mixtures such as liposomes, detergent, serum, and cell lysates. Thermophoresis is detected and quantified using attached or intrinsec fluorophores.The capillary format is used in the whole MST experiment, and only 4 μl sample is needed to complete the experiment.

During a MST experiment, an infrared laser is used to induce a local temperature difference, resulting in a local change in molecular concentration (depletion or enrichment). Changes in the thermophoresis of fluorescent molecule (TARGET) at a constant concentration, within a stable buffer environment, at different concentrations of a non-fluorescent molecule (LIGAND), can be used to  precisely quantify binding and determine affinity. The applications range from small-molecule binding events to protein-protein interactions and interactions of multi-protein complexes.

Highlights

• Easy to handle
• Immobilization free, in-solution measurements
• Low sample consumption (<10 μl /ligand concentration)
• Short experiment time (K_D in about 10 min)
• Very sensitive (KD range from pM-mM)
• No limitation of molecular size or mass (100 Da -1 MDa)
• Wide size range for interactants (from ions to MDa complexes)
• Wide range of buffers
• Measurement in complex mixtures (cell lysates, serum, detergents, liposomes)
• Wide temperature range (20-45 °C)
• Broad application range

Required Supplies

• MST Capillaries
  • Monolith NT.115 Standard Treated Capillaries, MO-K022
  • Monolith NT.115 MST Premium Coated Capillaries, MO-K025

Premium capillaries feature an MST-optimized polymer surface coating which suppresses biomolecule adsorption for best results, but standard Capillaries also work well for many assays. (esto como una nota dentro de MST capillaries. Puede ser en tamaño más pequeño)

• Labeling Kits
  • Protein Labeling Kit RED-NHS 2nd Generation (Amine Reactive), MO-L01  
  • His-Tag Labeling Kit RED-tris-NTA 2nd Generation, MO-L018   

Manuals

• MST getting started guide   
• Control software

Dedicated to running and analyzing MST experiments. It guides the user step-by-step through planning, setup and execution of experiments and helps to evaluate and analyze measured data. 

• Affinity Analysis software

Used for analysis and evaluation of MST data. 

References

• Duhr S, Braun D. Why molecules move along a temperature gradient. Proc Natl Acad Sci U S A. 2006; 103:19678-19682. https://doi.org/10.1073/pnas.060387310
• Jerabek-Willemsen M, Wienken CJ, Braun D, Baaske P, Duhr S. Molecular interaction studies using microscale thermophoresis. Assay Drug Dev Technol. 2011; 9:342-353. https://doi.org/10.1089/adt.2011.0380
• Jerabek-Willemsen M , André T , Wanner R , Marie Roth H , Duhr S, Baaske P , Breitsprecher D. MicroScale Thermophoresis: Interaction analysis and beyond. Journal of Molecular Structure 2014; 1077:101-113 http://dx.doi.org/10.1016/j.molstruc.2014.03.009
• Langer A, Bartoschik T, Cehlar O, Duhr S, Baaske P, Streicher W. A New Spectral Shift-Based Method to Characterize Molecular Interactions. Assay Drug Dev Technol. 2022; 20:83-94. https://doi.org/10.1089/adt.2021.133 Erratum in: Assay Drug Dev Technol. 2022; 20:136. https://doi.org/10.1089/adt.2021.133.correx
• Seidel SA, Wienken CJ, Geissler S, Jerabek-Willemsen M, Duhr S, Reiter A, Trauner D, Braun D, Baaske P. Label-free microscale thermophoresis discriminates sites and affinity of protein-ligand binding. Angew Chem Int Ed Engl. 2012;51:10656-10659. https://doi.org/10.1002/anie.201204268
• Seidel SA, Dijkman PM, Lea WA, van den Bogaart G, Jerabek-Willemsen M, Lazic A, Joseph JS, Srinivasan P, Baaske P, Simeonov A, Katritch I, Melo FA, Ladbury JE, Schreiber G, Watts A, Braun D, Duhr S. Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions. Methods. 2013;59:301-15. https://doi.org/10.1016/j.ymeth.2012.12.005
• Wienken CJ, Baaske P, Rothbauer U, Braun D, Duhr S. Protein-binding assays in biological liquids using microscale thermophoresis. Nat Commun. 2010;1:100. doi: 10.1038/ncomms1093.