MST - MicroScale Thermophoresis.
MST measures the directed movement of molecules along a temperature gradient.
When to use MST.
Thermophoresis—also known as the Soret effect—is the phenomenon where molecules migrate in response to a temperature difference. The magnitude and direction of this movement depend on a molecule’s size, charge, and hydration shell. When a fluorescently labeled target binds to a ligand, these properties change, altering the molecule’s thermophoretic behavior and providing a quantifiable readout of binding.
MST is particularly valuable when you need to:
When to use MST.
Thermophoresis—also known as the Soret effect—is the phenomenon where molecules migrate in response to a temperature difference. The magnitude and direction of this movement depend on a molecule’s size, charge, and hydration shell. When a fluorescently labeled target binds to a ligand, these properties change, altering the molecule’s thermophoretic behavior and providing a quantifiable readout of binding.
MST is particularly valuable when you need to:
- Measure challenging interactions that fail in other methods (membrane proteins, intrinsically disordered proteins, weak fragments)
- Work in complex buffers or native conditions (detergents, high salt, serum)
- Detect aggregation alongside binding measurements
- Measure a wide range of affinities from pM to mM in a single assay format
- Use minimal sample (low microliter volumes, nanomolar concentrations)
MST requires fluorescent labeling of one binding partner. This can be done with covalent dyes, fluorescent proteins, or fluorescent fusion tags.
The science behind MST.
Molecules move in temperature gradients
When you create a temperature gradient in a solution, molecules don’t just sit still. They move, either toward warmer regions or cooler regions, depending on their physical properties. This directed motion is thermophoresis.
The thermophoretic mobility of a molecule is determined by three main factors:
Size: Larger molecules experience different thermophoretic forces than smaller ones
Charge: The distribution of charge across the molecule affects how it interacts with the temperature gradient
Hydration shell: The layer of ordered water molecules surrounding the protein influences its movement
When any of these properties change—such as when a protein binds to a ligand—the thermophoretic mobility changes too.
An infrared laser creates a localized temperature gradient
In an MST experiment, an infrared (IR) laser heats a small region of the sample, creating a temperature difference of a few degrees Celsius. This localized heating establishes a temperature gradient.
Fluorescently labeled molecules in the heated region respond to this gradient by moving. Their movement is tracked by monitoring the fluorescence intensity in the heated zone. As molecules move away from (or toward) the heated region, the fluorescence intensity changes.
The MST signal has two components
When you turn on the IR laser, two things happen to the fluorescence signal:
- Temperature-Related Intensity Change (TRIC)
The moment the laser turns on, there’s an immediate change in fluorescence intensity. This is because most fluorophores are temperature-sensitive—their quantum yield changes with temperature. This initial jump is the TRIC component.
- Thermophoretic movement
Over the next few seconds, molecules move along the temperature gradient. If they move out of the observation volume, fluorescence decreases. If they move into it, fluorescence increases. This slower change is the thermophoresis component.
Both components are sensitive to binding. Either can be used to generate binding curves, though the thermophoresis signal often provides better separation between bound and unbound states.
How binding changes the MST signal
When a labeled target binds to a ligand, the resulting complex has different physical properties than the unbound target:
- The complex is larger (increased size)
- Charge distribution changes (ligand contributes its own charges)
- The hydration shell reorganizes (different surface exposed to solvent)
These changes alter how the complex responds to the temperature gradient. By measuring the MST signal across a dilution series of ligand, you can detect binding and quantify affinity.
How it works.
How it works.
MST detects ligand-induced aggregation
One of MST's unique strengths is its ability to detect ligand-induced aggregation. Some ligands—particularly hydrophobic small molecules or fragments—cause the target protein to aggregate rather than bind specifically.
Aggregation produces characteristic changes in the MST signal. Instead of a smooth binding curve, you see irregular behavior, often with a sharp increase or decrease in signal at higher ligand concentrations. This aggregation signature helps distinguish true binders from problematic compounds that cause non-specific aggregation.
Identifying aggregators is critical in drug discovery because they can produce false positives in other assays. MST's ability to flag these compounds saves time and resources by preventing aggregation-prone molecules from advancing in the pipeline.
Better show than tell. See how DLS generates information about your sample.
Measuring binding affinity with MST.
In an MST experiment, you prepare a series of samples containing:
- A constant concentration of fluorescently labeled target
- A dilution series of ligand (typically 16 concentrations spanning several orders of magnitude)
Each sample is loaded into a glass capillary and subjected to a brief IR laser pulse (typically 20-30 seconds). The fluorescence is recorded before, during, and after heating.
The normalized fluorescence (Fnorm) is calculated as the ratio of fluorescence during thermophoresis to initial fluorescence.
Plotting Fnorm against ligand concentration produces a dose-response curve. The dissociation constant (Kd) is determined by fitting this curve to a binding model based on the law of mass action.
The Kd represents the ligand concentration at which 50% of the target molecules are bound. Lower Kd values indicate tighter binding (higher affinity).
“NanoTemper helps us turn challenging biophysical tasks into routine workflows. Their intuitive solutions give us reliable data faster, so our teams can focus on advancing drug candidates.”
Join the Newsletter
Get email updates. Your edge in biophysics. Subscribe today.
Our latest research in MicroScale Thermophoresis.
Frequently Asked Questions
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer tellus tellus, ornare a felis quis, semper tempus elit. Donec in facilisis magna.
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer tellus tellus, ornare a felis quis, semper tempus elit. Donec in facilisis magna.
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer tellus tellus, ornare a felis quis, semper tempus elit. Donec in facilisis magna.
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer tellus tellus, ornare a felis quis, semper tempus elit. Donec in facilisis magna.
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer tellus tellus, ornare a felis quis, semper tempus elit. Donec in facilisis magna.