Similar to previously published methods, the thermal stability of OVCA2, variants of OVCA2, and FSH1 was determined using differential scanning fluorimetry (DSF).[36 (link), 60 ] Proteins (0.3 mg/mL) were diluted in at least triplicate in PBS containing a 1:250 dilution of SYPRO Orange (ThermoFischer Scientific). The samples were heated from 15°C to 85°C at 1.0°C/min in a thermocycler (Bio-rad C1000 Thermocycler with CFX96 Real-time System, Hercules, CA) and the change in SYPRO Orange fluorescence followed over time ((λex = 450–490 nm, λem = 610–650 nm). The melting temperature (Tm) was determined by plotting the first derivative of fluorescence versus temperature and finding the temperature at the midpoint of the transition. As in previous analyses,[36 (link), 42 (link), 61 (link)] all graphs were normalized so that minimum fluorescence was set to 0 and maximum fluorescence set to 1.
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