Bace 1 activity assay kit

BACE-1 activities were tested using a BACE-1 activity assay kit (Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer’s instructions. The fluorescence signal amplification is detected subsequent to substrate cleavage by BACE-1 in the assay, which is founded upon the fluorescence resonance energy transfer (FRET) method.

Also, a β-secretase (BACE1) activity assay kit was purchased from Sigma-Aldrich (catalog number CS0010) and was used as described previously [26 (link)]. In this experiment, an enhancement of the fluorescence signal after substrate cleavage via BACE1 was observed [27 (link)]. The concentrations of compounds were the same as in the assay with AChE. The assay was performed using a black 96-well microplate. Fluorescence was measured on an Infinite M200 fluorescence spectrophotometer (TECAN, Männedorf, Switzerland) (ex. 320 nm; em. 405 nm) in triplicate experiments with a negative control (no enzyme) and a positive control (enzyme activity supplied). Secretase activity correlated with fluorescence value. Its 50% decrease was calculated as IC₅₀ (µM).

LC-MS grade water, methanol, acetonitrile, formic acid, Zorbex UPLC C₁₈ Silica Column, Agilent 6520 QTOF instrument, acetylcholinesterase from electric eel, butyrylcholinesterase from equine serum, amyloid beta protein (Link biotech), thioflavin T, Congo red dye, reserpine, and ajmalicine were purchased from Sigma Aldrich, India. MAO-B inhibition kit by Biovision. BACE-1 activity assay kit by Sigma Aldrich. RPMI 1640 medium, FBS, PBS, and other tissue culture grade chemicals were purchased from Himedia, India. PC12 (rat pheochromocytoma) cell line was obtained from NCCS, Pune, India. All other reagents used were of analytical grade. All preparations used in tissue culture experiments were filtered through 0.45 µm Axiva 25 mm CA filter. Ethical approval was obtained from GGS Indrapratha University for all the cell culture work done in proper confined facilities.