MicroScale Thermophoresis | MST
MicroScale Thermophoresis (MST) is a novel method that enables the quantitative analysis of molecular interactions in solution on the microliter scale with high sensitivity. The technique is based on the movement of molecules in temperature gradients, a physical effect called thermophoresis. Usually, a depletion or accumulation of molecules in an area of elevated temperature is observed.
This directed movement of molecules depends on their molecular size, charge, and hydration shell. Binding of a ligand to a target molecule leads to the change of at least one of these parameters and therefore to an altered thermophoretic movement of the target-ligand complex compared to the single molecules alone.
MST monitors the thermophoresis of a target molecule supplied with different amounts of ligand and uses this information to quantify binding parameters of the molecular interaction. Therefore MST can be used for the analysis of almost any kind of molecular interaction or modification of small molecules, proteins, peptides, DNA, sugars, or molecular complexes.
The figure above gives an overview of the technical setup of MST. (A) An infrared-laser (IR-laser) is used to generate a precise focal temperature gradient within a glass capillary, which is filled with the reaction sample comprising fluorescent target molecules and their ligand molecules. The temperature of the aqueous solution in the laser spot is raised by ∆T = 2-5 K. This temperature gradient induces the thermophoretic movement of the molecules in the capillaries. The target molecules within the infrared laser focus, are monitored by their fluorescence (intrinsic or labeled), and their motion along the temperature gradient is recorded. (B) Initially, the fluorescence in the sample is detected in the absence of a temperature gradient to ensure homogeneity of the sample. After 5 seconds, the IR-laser is activated leading to the establishment of the temperature gradient. This causes an initial steep drop of the fluorescence signal – the so-called Temperature- or T-Jump – which reflects the temperature dependence of the fluorophore quantum yield. After the T-Jump, a slower thermophoresis-driven depletion of fluorophores occurs. Once the IR-laser is deactivated, a reverse T-Jump and subsequent backdiffusion of fluorescent molecules can be observed. (C) Since the thermophoresis is highly sensitive towards changes in molecular properties, a ligand-against-target titration series can be made. From that the equilibrium dissociation constant KD can be determined. For this, a serial dilution of the ligand is prepared, mixed with a constant concentration of labeled target molecule, loaded into capillaries and analyzed in the instrument by subsequent scanning of each capillary. The changes in thermophoresis are then plotted and used to derive the binding constant. The results of a typical binding experiment are illustrated in (D).
MST allows for the monitoring of either fluorescently-labeled molecules or intrinsically fluorescent molecule /(such as proteins; the latter would be a truly label-free measurement). MST measurements are possible in any kind of buffers, even in serum, plasma, cell lysate, urine, mucus, or other environmental matrices. Data generation is fast and precise and the data output is comparable to other biophysical methods.
- Hight-throughput target-ligand interaction screenings
- Steady-state binding assay
- Steady-state binding assay in biological liquids
- Sandwich assays (1 target, 2 ligands)
- Competition assays
- Binding assays with multiple binding partners
Compatible Fluorescent Dyes
In order to perform fluorescence-based measurements, one of the binding partners has to be fluorescently labeled. This enables the tracking of its molecular movement along the temperature gradient. Intrinsically fluorescent proteins can be analyzed without the need for further fluorescent labeling. The same goes for any molecule that possess intrinsic fluorescence in the wavelength regions outlined in the table below.
Alternatively, molecules with no intrinsic fluorescence can be labeled with fluorophors. Most commonly, the labels listed in the table below are used. 2bind offers the labeling of your target protein with all possible dyes. Another option is to fuse a potential target protein with an intrinsically fluorescent protein fluorophore such as GFP, RFP or the like.
In the case of DNA or RNA target molecules direct covalent linkage of fluorophores (e.g. Cy5) has proven well.
The following table gives an overview over the most commonly used fluorophores in MST. Please keep in mind that, in principle, every fluorophore can be used as long as its exitation and emission wavelength ranges match the ones of the fluorophores listed here.
|Fluorophor||Excitation (nm)||Emission (nm)|
- Low sample consumption (minimum of only 6 µl is required per sample)
- Free choice of assay buffers (also biological liquids possible such as serum or cell lysate)
- Very short analysis time (short analysis time enables high throughput)
- Real-time quality controls (online aggregation, precipitation, and sticking controls)
- Wide temperature range (analysis possible from 20°C to 45°C)
- No immobilization required (measurement is done truly in solution)
- Wide concentration range (affinities can be analyzed in the pM-mM range)
- Wide molecule size range (from 100 Da to 1 MDa)
FAQ – General
What kinds of molecular interactions can I measure?
What information do I get from an MST measurement?
Our sophisticated technology is not only able to determine affinities, but you can also assess other physical parameters such as stoichiometry, aggregation, precipitation, enthalpy (van’t Hoff plot), slow enzyme kinetics, and oligomerization.