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MicroScale Thermophoresis (MST)

MicroScale Thermophoresis (MST) is an immobilization-free technology for measuring biomolecular interactions with a wide range of affinities (pM-mM). The MST instrument detects the motion of fluorescent molecules along a microscopic temperature gradient, which reflects changes in the molecular hydration shell, charge or size. Since one or all of these parameters changes with virtually every binding event, a wide range of biomolecules can be measured from ions and small molecule fragments to large macromolecular complexes in very small volumes (<10 ul) in a wide range of standard buffers and complex mixtures including liposomes, detergent, serum, and cell lysates.

 

The CMI has a Monolith NT.115pico from NanoTemper Technologies.

For information on access fees, policies and getting started at the CMI, see the CMI Access Page.

Monolith NT.115pico Instrument Detectors

Pico RED detector

  • excitation wavelength: 600-650 nm
    • eg. AlexaFluor647, NT647, Cy5
  • fluorophore concentration  ≥ 50 pM
  • Kd range: pM - mM
Nano BLUE detector         
  • excitation wavelength 460-490 nm
    • eg. fluorescein, AlexaFluor488, NT495, GFP
  • fluorphore concentration ≥ 5 nM
  • Kd range: nM - mM

Monolith NT.115 BLUE/RED Excitation/Emission Filters

Key Features

  • fast measurement: Kd in about 10 min
  • wide Kd range from pM/nM to mM range
  • low sample consumption: sample volume (<10 µl per concentration)
  • immobilization free, in-solution measurements
  • measurements in complex mixtures (cell lysates, serum, detergents, liposomes)
  • wide size range for interactants (from ions to MDa complexes)

MST Theory

MicroScale Thermophoresis (MST) detects the motion of fluorescent molecules along a microscopic temperature gradient, which reflects changes in the molecular hydration shell, charge or size. A local temperature difference ΔT, induced by an infrared laser, leads to a local change in molecule concentration (depletion or enrichment), quantified by the Soret coefficient ST, chot/ccold=exp(-STΔT). This directed movement of molecules along a temperature gradient is termed "thermophoresis". Changes in the thermophoresis of a fixed concentration of fluorescent molecules in a constant buffer reflect changes in the size, charge or solvation entropy of the fluorescent molecules due to binding of the non-fluorescent partner (the titrant). Measuring this behavior at different concentrations of titrant allow quantification of the binding and determination of affinity.

Required Supplies

  • fluorescent target sample and non-fluorescent ligand sample and matched buffer
  • MST capillaries (see below)
  • 0.2 ml tubes for sample preparation (provided by CMI)
  • pipetors and tips for liquid handing

NanoTemper MST Capillaries

  • Monolith NT.115 Standard Treated Capillaries, MO-K002 (For now, the CMI has Standard capillaries for use.)
  • Monolith NT.115 MST Premium Coated Capillaries, MO-K005
  • Monolith NT.115 Hydrophobic Capillaries, MO-K003

 Labeling Kits

  • NanoTemper Protein Labeling Kit RED-NHS (Amine Reactive), MO-L001
  • NanoTemper Protein Labeling Kit RED-MALEIMIDE (Cys Reactive), MO-L004
  • NanoTemper His-Tag Labeling Kit RED-tris-NTA, MO-L008 

All MST experiments are setup with one fluorescently labeled molecule (the Target) at a fixed concentration that is mixed with various concentrations of a non-fluorescent molecule (the Ligand).

Samples

Target (the fluorescent molecule)

  • ~200 µl/titration at >2X working concentration (5-20 µM unlabeled protein to use with a labeling kit)
  • RED detector:
    • recommended stock concentration of labeled Target: >10 nM
    • recommended working concentration: 5 nM (for Kd > nM)
    • minimal working concentration: ≥ 50 pM (used for Kd in pM range)
  • BLUE detector:
    • recommended stock concentration of labeled Target: > 20 nM
    • recommended working concentration: 20 nM
    • minimal working concentration: ≥ 5 nM

Ligand (the non-fluorescent binding partner)

  • ~20 µl/titration, at 2X working concentration (bring the highest stock concentration available for an unknown Kd)
  • recommended stock concentration ≥ 100X the expected Kd
  • recommended working concentration ≥ 50X Kd

Reaction Buffers

Many buffers are compatible with MST, so it's usually a good idea to start with a buffer system in which your binding partners are well behaved. 

  • Addition of 0.05% Tween 20 or other detergent is usually required to prevent sticking to the capillaries.
  • Each capillary should be prepared with identically matched buffer.
    • Assay buffer (with detergent) is used to dilute the fluorescent molecule to 2X,
    • Ligand buffer, which should match the high concentration of ligand, is used to dilute the ligand. 
  • 0.5-1mg/ml BSA can also be used to minimize non-specific binding.  
  • Buffer cannot be opaque.
  • High viscosity samples may be hard to fill (up to 10% glycerol is fine).