Thioflavin t

Samples were normalized by the concentration of insoluble protein. Fluorescence readings of the insoluble fractions were performed in the presence of 5 μM thioflavin T (Sigma), using a Hitachi F7000 Fluorescence Spectrophotometer (excitation 435 nm, emission 485 nm). Fluorescence signal of the samples in absence of thioflavin T and a thioflavin T blank were used as background. For thioflavin T experiments in protein degraded samples, we subjected the insoluble fractions to a strong digestion treatment with 1 mg/ml proteinase K (PK, Sigma) for 1 h at 37°C, digestion was stopped with 5 mM phenylmethylsulfonyl fluoride (PMSF, Sigma). Digested products were mixed with 5 μM thioflavin T (Sigma), and fluorescence was determined as stated above.

Bovine insulin, thioflavin-T (ThT), all buffer chemicals and all solvents were purchased from Sigma-Aldrich. Solvents were of chromatography grade. Bovine insulin was purchased in powdered form (Product No I5500). thioflavin-T (ThT) was first dissolved in tetrahydrofuran and re-crystallized to remove impurities and then dissolved in distilled water from a milli-Q water system (Merck Millipore, Darmstadt, Germany). The solution was filtered through a 0.2 µm syringe filter to remove undissolved particulates. The ThT concentration was determined by absorption on a Cary 50 absorption spectrophotometer (Agilent Technologies, Santa Clara, California, US), using an extinction coefficient of 36,000 M⁻¹ cm⁻¹ at 412 nm. Insulin stock solutions were prepared by dissolving powdered bovine insulin in a small amount of 100 mM HCl to ~ 15 mg/mL, followed by dilution into one of the following buffers: 100 mM glycine–HCl, pH 2.2; citrate–phosphate buffer prepared from 100 mM citrate and 200 mM Na₂HPO₄ (McIlvaine 1921 ), spanning from pH 3.0 to 7.0, or 200 mM ammonium acetate, pH 4.0. The insulin concentration was determined by absorption, using an extinction coefficient of ε₂₈₀ = 5,800 M⁻¹ cm⁻¹.