Protein thermal shift assay
SYPRO Orange Protein Gel Stain 5000× (Thermo Scientific, United States) was diluted with protein buffer solution (20 mM Tris-HCl at pH 8.0 with 287 mM NaCl) to 200×. A 30 μg portion of protein (5.0 mg/mL) was mixed with BWX and incubated on ice for 60 minutes, then centrifuged at 14000 rpm/ minutes for 10 minutes. After centrifugation, the protein was evenly mixed with 2.5 μL 200× SYPRO Orange, and the total volume was 20 μL. The mixture was heated from 25 °C to 99 °C at a rate of 0.2 °C/minute, and each sample was prepared in triplicate. An Applied Biosystems real-time PCR instrument (7500 Fast, Thermo Scientific, United States) was used to determine the protein denaturation temperature (exposure temperature of hydrophobic amino acid residues), and the results were generated using Origin software (Niesen, Berglund, & Vedadi, 2007).