(ThermoFisher), according to the manufacturer’s protocol, using 0.25
mg/mL purified BOK and BAK. Briefly, for each protein sample, a 90 μL
MasterMix was prepared by using the buffer and the fluorescent dye ROX,
vortexed at low speed, and 4 aliquots of 20 μL each were dispensed in
4 individual wells of Microamp Fast Optical 96-well reaction plates
(ThermoFisher) for analysis in an Applied Biosystems 7500 thermal cycler
(ThermoFisher). The temperature gradient was 25°C–99°C
at a rate of 1°C/min. Data were analyzed with Protein Thermal Shift
Software v1.3 (ThermoFisher) to determine the melting temperature, using the
first derivative and Boltzmann equation non-linear regression. Dye-only
controls were included in each plate. Experiments were performed at least 2
times in quadruplicates for each protein sample at the 2 protein
concentrations. More recently, TSA assays were performed in 20 mM NaOAc pH
5.0 or 20 mM HEPES pH 6.8 and 150 mM NaCl, and 384-well plates in an Applied
Biosystems 7900HT Fast Real-Time PCR System (ThermoFisher). Final data
analysis and presentation were done in Microsoft Excel and GraphPad Prism v7
(GraphPad Software).