Bace 1 activity detection kit

Quantification of Aβ peptide and BACE-1 activity in fly brain were conducted as formerly detailed without modification [50 (link)]. In brief, for quantification of Aβ_1–42 peptide, 25 to 30 fly heads were homogenized in a 5 M guanidine-HCl containing protease Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA, USA) and centrifuged at 12,000× g for 15 min at 4 °C. The supernatant was then subjected to human Aβ42 ELISA kit (Life Technologies, Invitrogen, Carlsbad, CA, USA). Concentration of Aβ was determined by comparing with standard Aβ_1–42. For BACE-1 activity, the same numbers of fly heads were homogenized in T-PER™ Tissue Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA) without protease inhibitor. The lysate was then subjected to BACE-1 activity detection kit (Sigma-Aldrich, St. Louis, MO, USA). One unit of BACE-1 activity means it hydrolyzes 1.0 picomole of 7-Methoxycoumarin-4-acetyl-[Asn⁶⁷⁰, Leu⁶⁷¹]- Amyloid β/A4 Precursor Protein 770 Fragment 667-676- (2,4-dinitrophenyl)Lys-Arg-Arg amide substrate per minute at pH 4.5 at 37 °C.

BSP was prepared from black sesame seeds (Sesamum indicum L.) as previously described [46 (link)]. Pepsin from porcine gastric mucosa, pancreatin from porcine pancreas, porcine bile extract, 4-hydroxy-3-methoxybenzoic acid (VA), 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, KOH, NaOH, Na₂S₂O₄, AChE from Electrophorus electricus (E.C.3.1.1.7, Type VI-S), acethylthiocholine iodide, 5,5′-dithiobis-2-nitrobenzoic acid (DTNB), BChE from equine serum (E.C.3.1.1.8), butyrylthiocholine iodide, thioflavin T, 1,1,1,3,3,3-hexafluoro-2-propanole (HFIP) and BACE1 activity detection kit were purchased from Sigma-Aldrich (Milan, Italy); synthetic Aβ 1-40 and 1-42 (human sequence) were from Bachem (Bubendorf, Switzerland).

This assay was performed according to BACE1 Activity Detection Kit protocols (Sigma, St. Louis, MO). Briefly, BACE1 enzyme and substrate were diluted in a Fluorescent Assay Buffer to produce 10 x working solutions [24 (link)]. The aptamer was also diluted into different concentrations in deionized water. This assay was performed in 96-well microplates using 100 μl, which comprised of 20 μl of substrate working solution, 2 μl of BACE1 working solution, and 5 μl of different aptamer concentrations. The reaction was allowed to proceed for four hours in the dark under a lid at 37°C, and was terminated by a stop solution. The product fluorescence before and after the reaction was measured by a VICTORTM X3 multi-label plate reader (PerkinElmer, Waltham, MA, USA) using 320 nm excitation and 405 nm emission wavelengths. The percentage of inhibition was calculated using the following equation: 100-(IFi/IFo×100), where IFi and IFo represent the BACE1 fluorescence intensities in the presence and absence of inhibitor, respectively. Inhibition curves were created by graphing the percentage of inhibition versus the logarithm of the inhibitor concentration using linear regression.