Ls50b luminescence spectrometer

The γ-secretase activity was assayed in vitro using an APP-CTF-derived intramolecularly quenched fluorescent peptide (Calbiochem) according to the manufacturer's instructions and as described [44] (link). Briefly, cellular membranes from N2a cells (SNX17 knockdown and control, expressing HA-ApoER2) were solubilized in CHAPSO buffer (50 mM Tris–HCl, 2 mM EDTA, protease inhibitors, 0.25% CHAPSO, pH 6.8), followed by incubation at 37°C for different times in 150 μL of assay buffer (50 mM Tris–HCl, protease inhibitors, 2 mM EDTA, 0.25% CHAPSO, pH 6.8, and 8 μM fluorescent APP-CTF-derived peptide). After incubation, the reaction mixture was centrifuged (16,100×g, 15 min) and the supernatant transferred to a 96-well plate. Fluorescence was measured using a PerkinElmer Luminescence spectrometer LS50B (excitation/emission at 350/440 nm). The specific γ-secretase activity was determined after subtracting the fluorescence obtained in the presence of DAPT (10 μM). Background fluorescence was calculated by separately incubating 50 μg of CHAPSO-solubilized P2 membranes and 8 μM APP-CTF-derived peptide with assay buffer for different times and mixing them just before fluorescence determination.

In ancestor and evolved lines, proteasome activity was quantified as described previously [19 (link)] in three replicates each. From the middle of the exponential phase, 2 × 10⁸ cells were collected, washed and frozen at -80°C. Cell pellets were resuspended in 350 μl of lysis buffer (10 mM Hepes, 10 mM KCl, 1.5 mM MgCl), two-thirds volume of glass beads and were disrupted using a Precellys disrupter (three cycles of 10 s at 5,000 rpm followed by 2 min on ice between cycles). Pellets were centrifuged for 5 min at 3,000 g followed by 10 min at 15,000 g. Protein extracts were quantified using the BCA protein quantification kit (Pierce). 100 μg of protein extracts were resuspended in assay buffer (10 mM Tris pH 8, 20 mM KCl, 5 mM MgCl) to a final volume of 100 μl and were incubated at 37°C for 15 min. The proteasome substrate N-SLLVY-MCA (Sigma) was added to a final concentration of 50 μM, and cells were incubated at 37°C during 60 min with agitation. Activity was measured using a Perkin Elmer Luminescence Spectrometer (LS 50B) at 365 nm (excitation) and 435 nm (emission).

Plasma concentration of thiobarbituric acid-reactive substances (TBARS) was determined using the spectrofluorometric method, optimized by Wasowicz et al. [31 (link)]. TBA-reactive compounds were extracted to butanol. The value of fluorescence of butanol layer was read at an excitation wavelength of 525 nm and an emission wavelength of 547 nm, using the PerkinElmer Luminescence Spectrometer LS50B (Norwalk, Ct, USA).

The fluorescence
of the optical probe, 8-Anilino-1-napthalenesulfonate (ANS), is highly
sensitive to the environment and can easily increase about 20 times
in a nonpolar or rigid environment.⁴⁴ Binding
of ANS to hydrophobic patches of amyloid fibrils results in a significant
increase in fluorescence intensity.^{45 (link)} For
validation of the presence of fibrils, ANS (10 μM) was supplemented
to aliquots of the samples of interest, before and after fibril formation.
The samples were excited at 395 nm, and the emission spectra were
recorded from 410 nm to 600 (PerkinElmer luminescence Spectrometer
LS-50B, UK).

Sodium dithionite was prepared as a 1M solution in ddH₂O and was used 100 mM to chemically reduce 10 mM NBDHEX in 20 μl 0.5% NH₃. After 5 min, 2 μl reaction was diluted to 200 μl in 67 mM potassium phosphate buffer (pH 7.5). NBDHEX fluorescence quenching was verified at 25°C in a Luminescence Spectrometer LS50B (Perkin–Elmer) whereas the UV-visible spectra was acquired at 25°C in a Multiskan Spectrum (Thermo Scientific) spectrophotometer in 0.5 ml quartz cuvettes. For the mass spectrometry analysis, the reaction mixture was diluted in 50% ethanol and 1% ammonia and directly infused in the LTQ mass spectrometer through a glass tip. The nanospray ionization in positive ion mode was allowed applying 1.5–1.6 kV. HV Potential. Full MS in the 150–350 m/z range were acquired; then MS3 fragmentation of the ions 280 and 250, derived from MS2 of 298 and 268, respectively, was induced.

MAA-BSA was generated by incubating 2 mg/mL of BSA with 100 mmol/L solution of MDA (molar mass: 72.0636 g/mol) in the presence of 200 mmol/L acetaldehyde in phosphate buffered saline (PBS) at pH 4.8 for 3.5 h at 37°C in a sealed polypropylene tube in a non-oxidizing atmosphere (argon gas). Unmodified BSA (2 mg/mL) was used as control. Other controls such as MDA- or acetaldehyde-BSA adducts were prepared under the same conditions and at similar concentrations of acetaldehyde or MDA alone when used in the generation of BSA-MAA. Following incubation, unbound MDA and acetaldehyde were removed by extensive dialysis against 3 changes of PBS (2L) for 24 h at 4°C then stored under argon in 50 ml polypropylene tubes at 4°C in the dark.
Unmodified BSA 2 mg/ml was used as control. Modification of BSA with MAA was assessed by the TNBS-test for measuring free amino groups in a protein and by measuring the specific MAA fluorescence spectrofluorometrically with Perkin Elmer Luminescence Spectrometer LS50B (Beaconsfield, England) at excitation wavelength 394 nm and emission wavelength 462 nm.