Rf 5301pc spectrophotometer

Transmission electron microscopy (TEM) was conducted using a Hitachi H-800 electron microscope at an acceleration voltage of 200 kV. High-resolution TEM (HRTEM) imaging was implemented by a JEM-2100F electron microscope at 200 kV. Fluorescence spectroscopy was performed using a Shimadzu RF-5301 PC spectrophotometer. UV-vis absorption spectra were obtained using a Shimadzu 3100 UV-vis spectrophotometer. Fourier transform infrared (FTIR) spectra were performed with a Nicolet AVATAR 360 FTIR instrument.

Calcein-entrapped large unilamellar vesicles (LUVs) composed of EYPC/EYPG (7∶3, w/w)and EYPC/CH (10∶1, w/w) were prepared by vortexing dried lipids in dye buffer solution (70 mM calcein, 10 mM Tris, 150 mM NaCl, 0.1 mM EDTA, pH 7.4). The suspension was subjected to ten freeze-thaw cycles in liquid nitrogen and extruded through polycarbonate filters (two stacked 100-nm pore-size filters) using a LiposoFast extruder (Avestin, Inc.; Ottawa, Canada). Untrapped calcein was removed by gel filtration on a Sephadex G-50 column. Passing through a Sephadex G-50 column usually resulted in an approximately 10-fold dilution of lipid vesicles. The eluted calcein-entrapped vesicles were diluted further to achieve the desired final lipid concentration of 64 µM for the experiments. Leakage of calcein from LUVs was monitored by measuring fluorescence intensity at an excitation wavelength of 490 nm and an emission wavelength of 520 nm on a model RF-5301PC spectrophotometer (Shimadzu; Kyoto, Japan). Vesicles dissolved in 10% Triton X-100 in Tris-buffer (20 µL) were used to establish 100% dye-release and the total volume of the assay was 2 mL. The percentage of dye-leakage caused by the peptides was calculated as follows:
where F is the fluorescence intensity of peptide-treated vesicles, and F₀ and F_t are the fluorescence intensities without peptides and with Triton X-100, respectively.

To assess the ATP-induced quench of the intrinsic Trp UV fluorescence of the FUS RRM domain, the spectra were measured with the excitation wavelength at 280 nm at 25 °C with a RF-5301 PC spectrophotometer (Shimadzu, Japan) on the sample of FUS RRM at 40 µM in 10 mM sodium phosphate buffer containing 150 mM NaCl (pH 6.8) in the presence of ATP at different concentrations.
ThT-binding assay followed the same protocol we previously used to monitor amyloid formation of FUS RRM^{14 (link)}. Briefly, a 2-mM ThT stock solution was prepared by dissolving ThT in milli-Q water and filtered through a 0.22-μm Millipore filter. The fresh working solution was prepared by diluting the stock solution into 10 mM sodium phosphate buffer containing 150 mM NaCl (pH 6.8) to reach a final ThT concentration of 50 μM. A 10-μL aliquot of each incubation solution or 10 μL aliquot of the incubation buffer as the control, was mixed with 130 μL of the ThT working solution in the dark for 10 min. The fluorescence emission spectra were acquired for three repeats with the excitation wavelength at 442 nm and slit widths: excitation at 5 nm and emission at 10 nm^{14 (link)}.

All the chemicals were purchased from Aladdin (Beijing, China), J&K (Beijing, China) or Sigma-Aldrich (St. Louis, MO, USA), unless specifically stated. All the reagents were commercially available and of analytical reagent grade. The other solvents were purified by fractional distillation. Prior to use, all solvents were purified by fractional distillation. Deionized water with a resistivity of 18.2 MΩ·cm was used from a Milli-Q water purification system (Millipore Corp., Billerica, MA, USA). NMR spectra were recorded by Bruker Avance 300 (Bruker Corp., Rheinstetten, Germany). Mass spectra were recorded on an Agilent 1100 LC-MS system (Agilent Technologies Inc., Foster, CA, USA). The preparation process of AIE dots was completed by a bath sonicator (Bransonic, CPX2800H-C, Emerson Electric Co., Ferguson, MO, USA). UV-Vis absorption spectra were recorded using a UV-2550 UV-Vis spectrophotometer (Shimadzu Corp., Kyoto, Japan). Fluorescence spectra were measured by a Shimadzu RF-5301PC spectrophotometer (Shimadzu Corp., Kyoto, Japan). Transmission electron microscopy (TEM) images of the AIE dots were taken by JEM-2100F at 200 kV (JEOL, Ltd., Kyoto, Japan). DLS measurement was performed using a Malvern ZetasizerNano ZS size analyzer (Malvern Panalytical Ltd., Malvern, UK) at room temperature.

The PC NDs were prepared using a reprecipitation method. First, Cur and PTX were dissolved in ethyl alcohol to provide solutions with concentrations of 2 mg/ml. Next, 0.4 ml of the PTX solution and 0.1 ml of the Cur solution were quickly added to 4.5 ml of deionized water, vortexed for 1 min and allowed to stand for 15 min to produce PC NDs. Finally, PC NDs were purified via ultrafiltration and collected via lyophilization, which provided a 4:1 weight ratio (PTX to Cur) after quantified by UV-vis method.
The morphology of PC NDs was inspected by a transmission electron microscope (JEOL, Ltd., Japan) and a scanning electron microscope (FESEM, S4800, Hitachi Co. Ltd., Tokyo, Japan). Fluorescence spectroscopy was performed using a Shimadzu RF-5301 PC spectrophotometer. UV–vis absorption spectra were obtained using a Shimadzu 3100 UV–vis spectrophotometer. Fourier transform infrared (FTIR) spectra were performed with a Nicolet AVATAR 360 FTIR instrument. X-ray powder diffraction (XRD) investigation was carried out on a Rigaku X-ray diffractometer using Cu Kα radiation. A Nano-ZS 90 Nanosizer (Malvern Instruments Ltd., Worcestershire, United Kingdom) was used to determine the size distribution and zeta potential of PC NDs.