Cary eclipse spectrofluorometer

Transmission
electron microscopy (TEM) images were obtained using a JEOL JEM-1400
analytical TEM with an accelerating voltage of 120 kV. The ultraviolet
(UV–vis) absorption spectra were recorded using a Shimadzu
UV-1900 spectrophotometer. A Cary Eclipse spectrofluorometer (Varian,
Palo Alto, CA, USA) was used to record the fluorescence spectra and
relative quantum yield (QY) values. Rhodamine B (RhB) was used as
a reference to measure the QY values of CDs.

Fluorescence measurements were performed at room temperature (~23°C) using a Cary Eclipse spectrofluorometer (Varian) as described before (Hu et al., 2008 (link)) with slight modifications.

Synthetic peptides with ≥95% purity were purchased from AlexoTech AB (Umeå, Sweden). The molecular weights of the peptides were verified using MS by the supplier. The peptides were dispersed in 10 mM HCl and insoluble material was removed by centrifugation (12000 × g for 15 min). The solubilities were estimated using dry weight measurements and absorbance at 280 nm (the latter only when the peptides contained tyrosine or tryptophan residues). Fibril formation was investigated by incubation at 50 °C for up to 2 days with or without agitation. Agitation at 500 rpm was achieved by a ThermoMixer C (Eppendorf). The lower temperature for these experiments compared to the fibrillation of SPI was chosen to avoid hydrolysis of the peptides. The presence of nanofibrils was assayed by ThT fluorescence and AFM. Samples for ThT were prepared by mixing 20 μl of the protein solution with 200 μl 50 μM ThT solution. Fluorescence was measured on a Varian Cary Eclipse Spectrofluorometer with excitation at 440 nm and emission spectra recorded between 460 and 600 nm. AFM was performed as described above. The peptide samples (only with agitation) were also investigated by CD, using the same instrument and protocol as described above.

ThT fluorescence of protein/fibril samples were measured on a Cary Eclipse Spectrofluorometer (Varian) using a quartz cuvette according to Nilsson.^{47 (link)} About 50 μL of protein/fibril solutions were mixed with 150 μL of ThT solution (0.005 mg mL⁻¹). The emission spectrum from 460 to 600 nm was obtained at an excitation wavelength of 440 nm; the excitation and emission slits were 5 and 10 nm, respectively. 10 mM HCl solution was deducted as a background.