MST measurements are possible either in a glass capillary setup (Monolith-series instruments, NT.Automated) or in a plate-based format (Dianthus). In both setups,  an infrared laser is used to generate a precise temperature gradient while an LED is used for the excitation of fluorescent molecules inside the glass capillaries or the plate wells. The capillaries or wells each contain a mix of fluorescent binding partner (usually called the “target” and non-fluorescent binding partner, which is titrated in a dilution series (usually called the “ligand”).

The sample fluorescence in each capillary or well is measured over time. After a certain time, the infrared laser is switched on and the sample is heated. Such resulting MST traces are recorded for each ligand concentration and plotted against time. The calculated Fnorm values from the MST traces (fluorescence in region F1 divided by fluorescence in region F0) are dose-dependent and can be well described by the law of mass action. A plot of Fnorm against ligand concentration then returns the dissociation constant K_D of the interaction.

 

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 of MicroScale Thermophoresis include:

• Hight-throughput target-ligand interaction screenings
• Steady-state binding affinity assay
• Steady-state binding affinity assay in biological liquids
• Sandwich assays (1 target, 2 ligands)
• Competition assays
• Binding affinity assays with multiple binding partners

For more information, please visit our Application Database. The different service areas can be found under MST Services.

MicroScale Thermophoresis (MST) relies on measuring the fluorescence of the studied molecules. Thus, one of the binding partners has to be fluorescent. Intrinsically fluorescent proteins can be analyzed without the need for further fluorescent labeling.

Depending on which MST Service you require, different fluorescent methods and/or labelings are possible.

Alternatively, molecules with no intrinsic fluorescence can be labeled with fluorophores. Most commonly, the labels listed in the table below are used. 2bind performs the labeling of your target protein for you 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 excitation and emission wavelength ranges match the ones of the fluorophores listed here.

Fluorophore	Excitation (nm)	Emission (nm)
BCECF	480	525
GFP	488	507
NT-495 (BLUE)	493	521
Fluorescein (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
NT-RED 2nd generation	650	670

 

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

Such label-free MST is often interesting for specific protein MST services, whose intrinsic fluorescence can be used as an alternatively to a fluorescent label.

MicroScale Thermophoresis (MST) offers a number of great advantages over other biophysical methods for determining the affinity of a molecular interaction. Follow this link for an overview over our possible services.

• 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

For a comparison of MST with other biophysical techniques, please refer to our Technology Comparison Guide.

Yes. Analyzing molecular interactions involving aptamers or other DNA- or RNA-molecules is no problem. Usually, MST measurements with aptamers are straight-forwards, because these DNA molecules can be synthesized directly with fluorescent labels at their 5′ or 3′ end. Most commonly, Cy5 or Alexa647 are used as aptamer or DNA fluorophores.

More information on this topic is available in our Aptamer Services.

MicroScale Thermophoresis (MST) is not only able to determine the affinity and binding strength of a molecular interaction, but  also allows for assessing other physical parameters such as stoichiometry, aggregation, precipitation, enthalpy (van’t Hoff plot), slow enzyme kinetics, and oligomerization.

For specialized kinetics measurements, consider Biolayer Interferometry. If you are interested in the thermodynamics of a molecular interaction, Isothermal Titration Calorimetry is an option. For an in-depth analysis of protein aggregation, protein stability and protein unfolding, take a look at the nanoDSF technique.

Our MST instruments detect blue, red, and green fluorescent dyes. We can measure GFP and RFP labeled proteins as well as substrates with a wide variety of chemically attached fluorophores. Please see above (“Compatible Fluorescent Dyes”) for more information on the specific dyes that can be used.

Yes, 2bind also offers analyzing the affinity of a molecular interaction without labeling of the molecules. The label-free MST 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).

MicroScale Thermophoresis measures equilibrium binding constants (steady-state affinity). 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.

An in-depth kinetics analysis of molecular interactions is possible with Biolayer Interferometry.

MicroScale Thermophoresis measures equilibrium binding constants (steady-state affinity) at a given temperature. By performing such experiments at different temperatures, the binding enthalpy can be derived from van’t Hoff analysis.

An in-depth thermodynamics analysis of a molecular interaction is possible with Isothermal Titration Calorimetry.

Yes, we are able to measure molecular interactions involving small molecules with the same high sensitivity and quality as for protein-protein, protein-DNA or protein-vesicle interactions. For more information on MicroScale Thermophoresis and small molecule please have a look at our Drug Discovery Services.

Dependent on the fluorophore (fluorescent label or intrinsic tryptophan fluorescence), between 1 – 100 nM of the labeled target molecule can be used. This target is titrated with the unlabeled small molecule compound in the range of ± factor 10 of the expected affinity (dissociation constant). For standard applications, 6 – 10 µl of sample material are used per data-point.

Yes, in general it is possible to work with the ionic strength that is best suited for your molecules. Most of the time, a buffer change is not required. For example, we have already successfully measured molecular interactions in a solution with a sodium chloride concentration of 1.5 M.

If you are not sure whether your sample protein really requires high ionic strength buffers, it is possible to investigate protein stability, thermal unfolding, and its aggregation behavior using nanoDSF. We offer a variety of Services in this respect.

Yes! We have measured protein binding to the 70S ribosome unit as well as the binding affinity to liposomes with a size of more than 100 nm.

Yes. We are able to analyze the affinity of a molecular interaction also in complex biological liquids like crude cell extract, serum, plasma, urine, sea water, etc. You don’t have to pass on sensitivity or precision when analyzing these types of samples.

If you are interested in the stabilty of your protein sample, this can be determined using our nanoDSF technology.