MicroScale Thermophoresis | MST

Overview

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.

Technology

MicroScale Thermophoresis (MST) is based on the observation that molecules move in temperature gradients. This movement, called “thermophoresis” is eponymous for this method. The thermophoresis of molecules depends on their size, charge, and hydration shell. Since at least one of these parameters is typically affected upon interaction of different molecules, the changes in the movement of a molecule along the temperature gradient can be used for the analysis of any kind of bi-molecular interaction.

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 Kcan 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.

Typical applications

  • 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)
BCECF 480 525
GFP 488 507
NT-495 (BLUE) 493 521
Fluorecein (FITC) 495 519
 Alexa488 495 519
 YFP 514 527
Alexa532 530 555
T AMRA 546 579
 Cy3 550 570
 RFP 555 584
 NT-547 (GREEN) 557 574
 Alexa546 560 672
 Cy5 649 670
 NT-647 (RED) 650 680
Alexa647 652 668

Label-free MST

Due to tryptophan residues in their amino acid sequence, proteins can be intrinsically fluorescent. The  intrinsic fluorescence of such protein can be used to monitor their thermophoretic movement. By that no interaction partner has to be modified and the MST assay is truly label-free.

In order to avoid background noise, it is important that only one binding partner is fluorescent in the tryptophan range.

Advantages

  • 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?

You can measure bi-molecular interactions between any kind of molecule: protein, RNA, DNA, small compounds, lipids, carbohydrates.

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.

What type of fluorescent dyes can I use?

The instruments at 2bind detect blue, red, and green fluorescent dyes. We can measure GFP and RFP labeled proteins and also substrates with a wide variety of chemically attached fluorophores. Please see above for more information on the specific dyes that can be used.

Is it possible to measure without labelling a molecule?

Yes, 2bind also offers measurements without labeling of the molecule. The label-free system measures best in pure samples and is exceptionally sensitive. In addition to tryptophan fluorescence, it can also work with any other molecule that shows a fluorescence in the range of 280 nm (ex) and 360 nm (em).

Can I measure binding kinetics with MicroScale Thermophoresis?

MicroScale Thermophoresis measures equilibrium binding constants. Typically samples are inserted in the instrument after the binding reaction reaches chemical equilibrium. Measurement of kinetics is possible if the equilibrium is not reached quickly. On average, the kinetics of reactions that take more than 20 minutes can be evaluated.

FAQ – Samples

Can I check for the quality of my samples?

Yes, for example an aggregation of your protein is easily detected by the MST instrument.

Is it possible to measure the binding of small molecules?

Yes, we are able to measure the binding of small molecules with the equally high sensitivity that is observed for protein-protein, protein-DNA or protein-vesicle interactions.

What are the required concentrations for small molecules?

Dependent on the fluorophore, about 1 nM – 100 nM of the labeled compound can be used. The labeled compound is titrated with the unlabeled compound in the range of ± factor 10 of the expected dissociation constant. For standard applications, 6 – 10 µl of sample material are filled into the capillary.

My protein is only stable at high ionic strength. Can I still test for binding with MST?

Yes, in general you can use the ionic strength which is best suited for your molecules, you do not have to change it for the measurement. (We already successfully measured binding events occurring in a solution with a sodium chloride concentration of 1.5 M).

I want to measure binding to a Mega-Dalton (MDa) molecule. Is this possible?

Yes, we measured protein binding to 70S ribosomes as well as protein binding to liposomes with a size of more than 100 nm.

FAQ – Assay Conditions

Are there any limitations to the buffers or additives I can use?

No, you can use any buffer and any additive as long as it does not negatively affect the stability or homogeneity of your sample.

Can I measure in biological liquids like serum or lysates?

Yes, we are able to measure in complex biological liquids like crude cell extract and serum. You don’t have to pass on sensitivity or precision when analyzing these types of samples.

What are the required concentrations for small molecules?

Dependent on the fluorophore, about 1 nM – 100 nM of the labeled compound can be used. The labeled compound is titrated with the unlabeled compound in the range of ± factor 10 of the expected dissociation constant. For standard applications, 6 – 10 µl of sample material are filled into the capillary.

My protein is only stable at high ionic strength. Can I still test for binding with MST?

Yes, in general you can use the ionic strength which is best suited for your molecules, you do not have to change it for the measurement. (We already successfully measured binding events occurring in a solution with a sodium chloride concentration of 1.5 M).

I want to measure binding to a Mega-Dalton (MDa) molecule. Is this possible?

Yes, we measured protein binding to 70S ribosomes as well as protein binding to liposomes with a size of more than 100 nm.
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