A solution containing 0.75 µM coumarin-30, 400 nM tubulin and 100 µM of a tested compound in BRB-80-T buffer was freshly prepared and incubated for 1 hour at room temperature, then centrifuged at 13,000 g at 4 °C for 10 min prior to each experiment. Next, the solution was introduced into a capillary. A tray with capillaries was scanned for initial fluorescence, illuminated using the 'blue' excitation spectrum at 100% power in the Monolith NT.115 instrument (NanoTemper Technologies) at room temperature. MST signals from the capillaries were then collected under the same illumination conditions.