The term MST-on time is used to describe two closely related, but different aspects of an MST measurement. MST-on time on the one hand signifies the time period during which the infrared (IR) laser is active and generating a local temperature change in the sample*. On the other hand, the MST-on time signifies the specific timepoint at which binding analysis is performed. As an example, in a typical measurement the IR-laser is active for 20 sec MST-on time but afterwards data analysis is performed with the F₁ cursor set to 1.5 sec MST-on time. Note that the analysis of a binding event is oftentimes best analyzed at MST-on times shorter than the experimentally measured MST-on time.

Figure 1 MST trace with indicated measured MST-on time and the MST-on time used for subsequent data analysis.

*The measured MST-on time is fixed to 20 s in experiments run with MO.Control software. MO.Screening Control software adjusts the measured MST-on time according to the MST power used (20 s for low MST power, 10 s for medium and high MST power). In Expert Mode (MO.Control) and Free Style Mode (MO.Screening Control), the MST-on time can be modified if desired.

Sample heating with an IR-laser

During the time the IR-laser is active, the temperature in the capillary is locally increased. The kinetics of the heating process are fast at first but then gradually decrease. The temperature applied to the sample therefore depends not only on the IR-laser intensity but also on the MST-on time. In other words, the shorter the MST-on time, the lower the overall increase in temperature. The change in temperature results in an alteration of the MST-signal of the sample (i.e fluorescence), which is specific for the bound and unbound state of a target molecule. By default, we recommend to perform assays at lower MST powers and to select short MST-on times for data evaluation, in order to minimize possible structural destabilization of biomolecules by the applied temperature. The automatic analysis algorithm of MO.Control, as well as the preset MST-on times in MO.Affinity Analysis and MO.Screening Analysis, already preferentially select for MST-on times with lowest possible temperature increase. Due to the local nature and high speed of the heating process, typically temperatures increases (ΔT) up to 10 K ensure reliable and reproducible results, without compromising the structural integrity of the sample.

Prolonged MST-on times

In some rare cases however, it can even be beneficial to make use of temperature-induced destabilization of your sample as target thermostability is oftentimes strongly influenced by a binding event. In these rare cases the MST-signal can be sensitive for destabilization of the target by increased temperature, an effect that is usually ligand dependent. Thus, binding-induced changes in the stability of a target molecule may result in dose-dependent MST-signals at late MST-on times. Consequently, for some interactions best results are obtained after prolonged MST-on times. Temperature-induced destabilization is oftentimes also the reason for fluorescence increases in MST traces at late MST-on times.

Effect of long MST-on times on K_d

To elucidate if a prolonged temperature increase also affects the thermodynamic equilibrium of an interaction and therefore also the measured affinity, one can compare the derived K_d values at different MST-on times. If the K_d does not change with the selected MST-on time, the temperature increase has no effect on the thermodynamic equilibrium of the interaction. If the K_d does however change with the MST-on time, we suggest selecting a lower MST-power or the shortest possible MST-on time. That will ensure selecting for the smallest possible temperature increase.

IR-laser induced temperatures

This table summarizes the MST-on times at different MST powers at which the temperature increase ΔT is less than 10 K. In the time ranges noted in the table, the temperature increase is time-dependent but always lower than 10 K. Therefore, an MST-on time of 1.5 seconds usually means a temperature increase of only few K. Note that the temperature increase is always less than 10 K at low MST power.