Thermal Shift Assay was performed to determine the thermal stability of the monobody mutants. The measurements were done using the Protein Thermal Shift™ Dye Kit (ThermoFisher Scientific, 4,461,146) and performed on a StepOne™ Real-Time PCR System (Applied Biosystems, ThermoFisher Scientific). Samples were measured in triplicates and contained 3 µg of protein in 1 × DPBS. A thermal profile from 25 °C to 95 °C with a ramp rate of 1% was acquired using StepOnePlus Software (Applied Biosystems, ThermoFisher Scientific) and analyzed using Protein Thermal Shift Software (Applied Biosystems, ThermoFisher Scientific).
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