Microscale Thermophoresis Assay Services

MST assays measure the motion of molecules along microscopic temperature gradients that changes upon ligand binding.

MST assays measure the ligand-target interactions directly in solution without the need for immobilization to a surface (immobilization-free technology). The technique requires low amounts of pure analyte samples and may be compatible with non-purified protein samples and is compatible with most buffers.

  • Binding between biomolecules of any type can be investigated including small-molecules, multi-protein complexes, DNA, liposomes, nano-particles and more
  • Fast lead time for assay development and affinity determination
  • Deliverable: dissociation constant KD (binding affinity)

MST Principle

When performing an MST experiment, a microscopic temperature gradient is induced by an infra-red laser, and the movement of fluorescent molecules away from the heated area is monitored. The movement varies depending on whether a ligand is bound to the molecule. The difference in motion is used to calculate binding affinity.

microscare thermophoresis (MST) Principle

Assay Details

Thermophoresis

example graph of microscale thermophoresis assay probing a compound to bind against a target protein

In a glass capillary, fluorescent molecules are allowed to interact with ligands in various concentrations. Upon laser beam treatment of a distinct area of the capillary, the intensity of fluorescence changes due to the movement of molecules away from the heated area (thermophoresis, also called thermo-migration), which differs when the ligand is bound. Temperature-induced changes in fluorophore properties may also be observed.

Example: BRD4

microscale thermophoresis graph example for compound testing for affinity binding to a target

Comparison of RVX208 binding to bromodomain 1 vs bromodomain 2 or BRD4.

MST measurements were performed for two bromodomains of BRD4 protein (BRD4-1 and BRD4-2) in the presence of variable concentrations of RVX-208 ligand. Determined KDs show a higher affinity of RVX-208 towards the second bromodomain of BRD4.

KD BD1: 2.2E-06M
KD BD2: 8.3E-08M

Screening Details

Instruments: Monolith NT.115 pico (NanoTemper) for the detection of binding affinities in the low pM range with the highest sensitivity.

Turnaround time: The study is typically completed within 2-3 weeks after receipt of materials. Projects are performed on a first-come-first-serve basis.

Sample requirements: Protein purity of 85% or more is required. The minimum protein amount is dependent on the storage buffer and labeling technique. The rough estimate is between 100-200 ug.
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Screening location: The assay is performed in Malvern, PA, USA.