MicroScale Thermophoresis (MST) is a technology pioneered by NanoTemper more than 10 years ago for measurements in solution from a mixture of target and ligand in the glass capillaries used in the Monolith instrument.
Since the change in fluorescence is dependent on the overall chemical environment, one needs to label the target molecule with a fluorophore sensitive to these changes. And any binding partner or ligand works, including proteins, nucleic acids, or small molecules.
The affinity between two molecules tells you how tightly they bind to each other. Affinity measurements are reported as the affinity constant, equilibrium dissociation constant, or Kd. The Kd and affinity are inversely related. The Kd value is related to the concentration of one of the binding partners and so the lower the Kd value — lower concentration expressed in molar values — the higher the affinity between the two molecules.
Scientists working in drug development use MST and Spectral Shift to tackle challenging interactions with a versatile tool perfect for working with a wide variety of projects.
The technologies are also used for the characterization of protein degraders’ binary and ternary complexes.
In an MST assay, the molecule you label with the fluorophore is called a target. The other binding partner — another protein, nucleic acid sequence, small molecule, or fragment — is called a ligand.
To calculate the Kd, a constant amount of the fluorescently labeled target is mixed with a dilution series of a ligand. The recorded changes in fluorescence are plotted against the logarithmic ligand concentration to build a binding curve. The Kd is determined from the binding curve using the law of mass action.
Additionally, MST gives you information about ligand-induced aggregation. Aggregation is very common, especially with insoluble fragments and small molecules with hydrophobic substitutions, and it’s often the reason why other technologies can’t determine binding affinities, but they never identify the cause of their failure.