ThermoFluor

The BACE1 ThermoFluor assay was performed in 384-well plates in a total volume of 4 μL in a buffer containing 50 mM NaOAc pH 5.4, 150 mM NaCl, 0.0005% Tween 20, 100 μM ANS, 1.6 μM mature BACE1 ectodomain protein and compound. Compound stock solutions in 100% DMSO were diluted 1/24 in the reaction, resulting in final compound concentrations of 2.1, 0.80, or 0.2 mM (depending on the initial compound stock concentration) and a final DMSO concentration of 4.2%. Thermal shift measurements were done in a ThermoFluor instrument from 25 to 85 °C in 1 °C increments. Fluorescence at every temperature was determined using a charge-coupled device camera and subsequent image analysis using proprietary software.