Molecular interactions that can be measured with MST
MST works with almost all classes of target molecules, as long as they are either intrinsically fluorescent or they can be labeled with a fluorescent dye. The variety of ligand molecules is even greater. The fluorescent target molecule is kept at a constant concentration and the non-fluorescent ligand molecule is titrated across a large concentration range.
If you are uncertain whether your type of interaction can be measured with MST or if you want to use the 2bind MST service just leave us a message:
What MST can tell
Single-dose screen
MST will tell you whether a target and ligand generally interact from a single-dose experiment.
Steady-State Affinity
Protein Aggregation
MST will tell you whether your protein samples aggregate or if aggregation is induced by ligand binding.
Ligand competition
MST will tell you competitive effects between multiple binders of your targets.
- Drug discovery (compound-based and fragment-based)
- Drug discovery (protein-targeted and RNA-targeted)
- Protein characterization
- Additive screening
- Enzyme-inhibitor screening
- Drug discovery (compound-based and fragment-based)
- Drug discovery (protein-targeted and RNA-targeted)
- Protein characterization
- Additive screening
- Enzyme-inhibitor screening
- Protein and enzyme quality control
- Buffer screening and optimization
- Protein storage optimization
- Ligand-induced aggregation testing
- Complex binding analysis
- Mode-of-action analysis
- Competitor screening
- PPI-disruption analysis
Advantages of MST
2bind MST Services
2bind offers a variety of services using the MicroScale Thermophoresis technology. Benefit from fast and precise analysis of molecular interactions.
Enjoy minimal sample consumption (just nM concentrations and µL volumes) and robust analysis methods. The 2bind MST services can be combined as you choose for all questions in drug discovery, antibody development, protein biophysics and analysis, as well as aptamer characterization.
MST Technology and FAQs
Overview
MicroScale Thermophoresis (MST Service) is a biophysical technique that measures the strength of interaction (affinity) between two molecules by detecting variations in fluorescence signal as a result of an IR-laser induced temperature change. The range of the variation in the fluorescence signal correlates with the binding of a ligand to the fluorescent target. By that it allows for quantitative analysis of molecular interactions in solution on the microliter scale with high sensitivity.
The MST signal is composed of two major factors: The TRIC effect and the thermophoresis. TRIC stands for Temperature-Related Intensity-Change and describes how the fluorescence intensity of a fluorophore depends on the local temperature of the solution. For the vast majority of fluorophores, the fluorescence intensity decreases with increasing temperature. Most importantly, however, the extent of the temperature dependence is strongly related to the chemical environment of the fluorophore. This environment is sensitively affected by the binding of a ligand molecule to the fluorescently labeled target molecule.
The second component of the MST-signal, the thermophoresis describes the movement of molecules along temperature gradients which results in a quantifiable change of the local concentration of the target molecules. As these target molecules are fluorescently labeled in MST, such concentration changes can be monitored simply by fluorescence measurements. 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.
In sum, both TRIC and thermophoresis contribute to the overall MST-signal, which is detected by fluorescence measurement. Due to this robust physical principle and direct monitoring, MST can be used for determining the affinity and binding strength of almost any kind of molecular interaction or modification of small molecules, proteins, peptides, DNA, sugars, or molecular complexes.
Technology
MicroScale Thermophoresis (MST Services) is based on the combined effect of temperature-related intensity-change of fluorescent molecules (TRIC) and their movement along temperature gradients (thermophoresis). Both the TRIC and the thermophoresis component of the MST-signal vary with three key parameters that are influenced by binding between the fluorescent target and the non-fluorescent ligand molecule: molecular size, molecular charge, as well as the hydration shell of the target molecule.
The figure above gives an overview of the technical setup of MST. (A) 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 capillary. The glass capillaries contain a mix of fluorescent binding partner and non-fluorescent binding partner, which is titrated in a dilution series. (B) A typical MST trace is shown: the sample fluorescence in one capillary is measured over time. After five seconds, the infrared laser is switched on, and the sample is heated (MST-on time). The Fnorm value is calculated from the ratio between the fluorescence after heating (F1) and before heating (F0). (C) MST traces are recorded in multiple capillaries containing constant amounts of fluorescent binding partner but varying amounts of non-fluorescent binding partner. (D) The calculated Fnorm from the MST traces is dose dependent and can be well described by the law of mass action. The fit returns the dissociation constant KD 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
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.
Compatible Fluorescent Dyes
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 |
Label-free MST
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.
Advantages
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.
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 molecule compounds, lipids, carbohydrates (See figure on top of this page).
Is it possible to analyze interactions with aptamers?
What information do I get from an MST measurement?
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.