Ls 55 fluorescence spectrophotometer

Once the docking studies yielded results that allowed us to select four compounds for experimental studies, we evaluated their effect on Aβ_1–42 pathological aggregation. For this purpose, lyophilized wild-type human Aβ_1–42 peptide (chloride salt) was purchased from Calbiochem (Mexico). HEPES sodium salt (>99.5% purity) and ThT were obtained from Sigma–Aldrich (Mexico). A freshly prepared Aβ_1–42 solution (50 μM in fresh MilliQ water) was incubated alone and in the presence of the selected compounds (100 μM) [26 (link)]. The samples were incubated at 37°C in a 0.5-cm path length quartz cell and stirred at 250 rpm. The increase in ThT fluorescence [31 (link)] was measured using a Perkin–Elmer LS-55 fluorescence spectrophotometer equipped with a water-jacketed cell holder for temperature control. The emission and excitation wavelengths were 445 and 480 nm, respectively. The fluorescence emission recordings were made using path-length quartz cuvettes. The buffer solution was 20 mM HEPES and 100 mM NaCl, pH 7.4, containing 3.3 μM ThT [26 (link)]. The inhibition of Aβ_1–42 pathological aggregation for compounds 5, 8, 14 and 19 was calculated after 24 h of incubation.

Absorption spectra and excitation/emission spectra of FPs were measured separately using a LAMBDA-25 UV/Vis spectrophotometer (Perkin Elmer, Waltham, USA) and a LS55 fluorescence spectrophotometer (Perkin Elmer, Waltham, USA) equipped with a red sensitive photomultiplier tube (Hamamatsu Photonics, Hamamatsu, Japan). Molar extinction coefficients were quantified using a alkali denaturation method [12 ,13 ]. In order to measure accurately the quantum yields, according to the description of optical dilution measurement [14 ], protein samples were first diluted in PBS (pH7.4) with UV absorption value in the range of 0.01–0.05. Fluorescence intensity of the mutant FP was compared with that of mNeptune (a quantum yield of 0.2). In order to facilitate the brightness comparisons between different FP variants, the brightness of all the proteins is converted to the relative brightness, which is relative to the brightness of EGFP (the product of quantum yield and extinction coefficient compared to the brightness of EGFP (53,000M^-1cm^-1×0.60) [15 (link)]).

All reagents (without further purification) were purchased from commercial suppliers. All experiments involving all compounds (1 and 2) were conducted in a PBS buffer solution (pH = 7.4, 10 mM, 50% (v/v) C₂H₅OH). Fluorescence spectra were obtained using a PerkinElmer LS 55 fluorescence spectrophotometer. UV-vis absorption spectra were measured on a UV-2550 spectrophotometer (SHIMADZU). ¹H and ¹³C-NMR spectra were determined using a Bruker FT-NMR spectrometer (600 MHz). Infrared spectra were recorded on an FT-IR infrared spectrometer (Nicolet 380). Fluorescence bio-imaging were finished using confocal laser scanning microscopy.