4.5. Protein Stability Determinations

Thermal stability tests were performed with the differential scanning fluorimetry technique also known as thermal displacement test (thermofluor). The experiment was performed on a 96-well plate in the Stratagene Mx3005P (Agilent Technologies, Waldbronn, Germany). In a final reaction volume of 40 μL, the proteins, at a final concentration of 0.5 mg/mL, were incubated in the presence of Sypro orange (Invitrogen) at a final 5-fold concentration, following the manufacturer’s instructions. The reaction was performed in 75 cycles of 60 s each, with an initial temperature of 25 °C and an increase of 1 °C per cycle. The emitted fluorescence was captured in each cycle and was plotted on Graphpad prism. To obtain the Tm, the curve data were fitted to the Boltzmann equation. Determinations were performed in 25 mM HEPES pH 7.0.