TTR unfolding assay using urea

Proteins unfolding of WT‐TTR, A97S‐TTR, T119M‐TTR, V30M‐TTR, and L55P‐TTR were assessed using urea denaturation assay.21 TTR sample solutions (0.1 mg/mL) were prepared by diluting 200 μL of 1.0 mg/mL TTR stock into 1.8 mL buffer, containing 10 mmol/L Na phosphate (pH7.5) with 100 mmol/L KCl, 1 mmol/L EDTA, 1 mmol/L DTT, 0.02 % sodium azide, and various concentrations of urea. The fluorescence emission ratio at 355 and 335 nm (F355/F335) of each sample was then monitored using a fluorescence spectrometer (Perkin Elmer LS‐55) after 96 h of incubation at room temperature. The excitation slits were set at 2.5 nm, while the emission slits were set at 3.5 nm. Excitation wavelength was fixed at 295 nm and the emission spectra were collected from 310 to 450 nm.