The labeling of the CTD of Hsp90 (Hsp90 CTD) was described before. For determination of the apparent K D value of Hsp90 CTD and the respective inhibitor, 10 µl of 50 nM labeled Hsp90 CTD in PBS containing 0.5 % BSA and 0.1 %Tween-20 were mixed with 10 µl of the respective inhibitor in different concentrations ranging from 15 nM to 500 µM and incubated for 15 min at RT in the dark. Thermophoresis of each mixture was induced at 1475 nm ± 15 nm and measured using the Monolith NT.115 (NanoTemper Technologies GmbH, München, Germany) . The measurement was repeated three times independently for each sample and each measurement was performed at 25 °C for 25 s at 70 % LED power and 40 % infrared laser power. The resulting fluorescence values were plotted against the concentration of the titrant and the apparent K D was calculated using the KD Fit formula Y = E + (A -E) / 2 * (T + x + K D -sqrt((T + x + K D )^2 -4 * T * x)) by GraphPad Prism software (GraphPad Software, Inc. La Jolla, USA). T, the concentration value for the labeled Hsp90 CTD, was set to 50 nM.