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).