MST is based on the quantifiable detection of a fluorescence change in a sample when a temperature change is applied. The fluorescence of a target molecule can be extrinsic or intrinsic (aromatic amino acids) and is altered in temperature gradients due to two distinct effects. On the one hand temperature related intensity change (TRIC), which describes the intrinsic property of fluorophores to change their fluorescence intensity as a function of temperature. The extent of the change in fluorescence intensity is affected by the chemical environment of the fluorescent probe, which can be altered in binding events due to conformational changes or proximity of ligands. On the other hand, MST is also based on the directed movement of molecules along temperature gradients, an effect termed thermophoresis. A spatial temperature difference ΔT leads to a change in molecule concentration in the region of elevated temperature, quantified by the Soret coefficient S:c/c = exp(-S ΔT). Both, TRIC and thermophoresis contribute to the recorded signal in MST measurements in the following way: ∂/∂T(cF)=c∂F/∂T+F∂c/∂T. The first term in this equation c∂F/∂T describes TRIC as a change in fluorescence intensity (F) as a function of temperature (T), whereas the second term F∂c/∂T describes thermophoresis as the change in particle concentration (c) as a function of temperature. Thermophoresis depends on the interface between molecule and solvent. Under constant buffer conditions, thermophoresis probes the size, charge and solvation entropy of the molecules. The thermophoresis of a fluorescently labeled molecule A typically differs significantly from the thermophoresis of a molecule-target complex AT due to size, charge and solvation entropy differences. This difference in the molecule's thermophoresis is used to quantify the binding in titration experiments under constant buffer conditions.
The thermophoretic movement of the fluorescently labelled molecule is measured by monitoring the fluorescence distribution F inside a capillary. The microscopic temperature gradient is generated by an IR-Laser, which is focused into the capillary and is strongly absorbed by water. The temperature of the aqueous solution in the laser spot is raised by ΔT=1-10 K. Before the IR-Laser is switched on a homogeneous fluorescence distribution F is observed inside the capillary. When the IR-Laser is switched on, two effects, occur on the same time-scale, contributing to the new fluorescence distribution F. The thermal relaxation induces a binding-dependent drop in the fluorescence of the dye due to its local environmental-dependent response to the temperature jump (TRIC). At the same time molecules typically move from the locally heated region to the outer cold regions. The local concentration of molecules decreases in the heated region until it reaches a steady-state distribution.
While the mass diffusion D dictates the kinetics of depletion, S determines the steady-state concentration ratio c/c=exp(-S ΔT) ≈ 1-S ΔT under a temperature increase ΔT. The normalized fluorescence F=F/F measures mainly this concentration ratio, in addition to TRIC ∂F/∂T. In the linear approximation we find: F=1+(∂F/∂T-S)ΔT. Due to the linearity of the fluorescence intensity and the thermophoretic depletion, the normalized fluorescence from the unbound molecule F(A) and the bound complex F(AT) superpose linearly. By denoting x the fraction of molecules bound to targets, the changing fluorescence signal during the titration of target T is given by: F=(1-x) F(A)+x F(AT).
Quantitative binding parameters are obtained by using a serial dilution of the binding substrate. By plotting F against the logarithm of the different concentrations of the dilution series, a sigmoidal binding curve is obtained. This binding curve can directly be fitted with the nonlinear solution of the  law of mass action, with the dissociation constant K as result.