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MicroScale Thermophoresis (MST)

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MST at the CMI

MicroScale Thermophoresis (MST) is an immobilization-free technology for measuring biomolecular interactions. 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 will change with virtually every binding event, a wide range of biomolecules can be measured, from ions and small molecule fragments to large macromolecular complexes, in small volumes (~20 μl), in a wide range of standard buffers and complex mixtures such as liposomes, detergent, serum, and cell lysates.

MST Theory

 

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

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 Supplies

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-K025

 Labeling Kits (optional)

  • NanoTemper Protein Labeling Kit RED-NHS 2nd Generation (Amine Reactive), MO-L011
  • NanoTemper Protein Labeling Kit RED-MALEIMIDE 2nd Generation (Cys Reactive). MO-L014
  • NanoTemper His-Tag Labeling Kit RED-tris-NTA 2nd Generation, MO-L018

 

Assay Buffers

  • Many buffers are compatible with MST. It’s usually a good idea to start with a buffer system in which your proteins are well behaved.
  • Addition of 0.05% Tween 20 or other detergent is usually required to prevent sticking of proteins 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 is used to dilute the ligand and should match the highest concentration of ligand
  • 0.5-1 mg/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).

Samples

  • All MST experiments are setup with one fluorescently-labeled molecule (the Target) at a fixed concentration mixed with various concentrations of a non-fluorescent molecule (the Ligand).
  • Concentration should be accurately measured
    • Errors in Target concentration can affect fluorescent signal and may affect the fit
    • Errors in the Ligand concentration will directly translate to errors in the KD
  • Protein aggregates will interfere with MST
    • Filter or centrifuge samples before use.
    • Assess protein heterogeneity via light scattering.
    • Purify protein samples with soluble aggregates by size-exclusion chromatography.

Target Sample (the fluorescent molecule)

  •  ~200 µL/titration at >2X working concentration
  • 5-20 µM unlabeled protein, if using a chemical labeling kit
  • RED detector:
    • 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:
    • Stock concentration of labeled Target: > 20 nM
    • Recommended working concentration: 20 nM
    • Minimal working concentration: ≥ 5 nM

Ligand Sample (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