The thermal stability of MmLipW and MmLipW variants was determined using differential scanning fluorimetry.45 MmLipW protein (0.30 mg/mL) was diluted in triplicate in PBS containing a 1:250 dilution of 5000× Sypro Orange dye (Invitrogen, Carlsbad, CA). The samples were heated from 15 °C to 90 °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 midpoint denaturation temperature (Tm) was determined by plotting the first derivative of fluorescence versus temperature and finding the temperature at the midpoint of the transition.
C1000 thermocycler with cfx96 real time system
Manufactured by Bio-Rad
The C1000 Thermocycler with CFX96 Real-time System is a laboratory instrument designed for performing polymerase chain reaction (PCR) experiments. It combines a thermal cycler with a real-time detection system, allowing for the amplification and quantification of DNA samples simultaneously. The device is capable of precisely controlling temperature and cycling conditions to facilitate the PCR process.
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2 protocols using c1000 thermocycler with cfx96 real time system
1
Thermal Stability Analysis of MmLipW
2
Thermal Stability Assessment of Proteins
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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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