β secretase activity fluorometric assay kit

Enzymatic activity of BACE1 (β-secretase) was determined using the β-secretase activity fluorometric assay kit (Biovision, Milpitas, CA, United States) according to the manufacturer’s instructions. Briefly, brain tissues were mixed with 2 volumes of ice-cold Extraction Buffer and homogenized on ice. After 10 min incubation on ice, the extracts were centrifuged at 10,000 × g for 5 m. Then, 50 μL supernatant was mixed with an equal volume of 2× reaction buffer and 2 μL substrate. The plates were kept in the dark at 37°C for 1 h, and the fluorescence was recorded at Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek, Winooski, VT, United States) with Ex. 340 nm and Em. 500 nm. BACE1 activity was expressed as relative fluorescence units per micrograms of protein sample.

The hippocampus samples were homogenized in ice-cold RIPA Lysis Buffer (Beyotime Biotech), supplemented with the PMSF, protease, and phosphatase inhibitor. The concentrations of Aβ 1–40 and Aβ 1–42 in the brain extracts were measured by using an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (R&D Systems, DAB142, DAB140B).
The β-site of the APP Cleaving Enzyme 1 (BACE1) activity in the fresh brain tissues was measured by using the β-Secretase Activity Fluorometric Assay Kit, according to the manufacturer’s instruction (Biovision, K360-100).

The assay was performed in a 96-well flat-bottom white plate using β-Secretase Activity Fluorometric Assay Kit (Biovision, CA, USA). To begin, HT22 cells were treated with HYP (20–80 μM) for 24 h and then collected by centrifugation with ice-cold extraction buffer added for homogenization. After centrifugation, 50 μL supernatant (cell lysate) was transferred to each well in the 96-well plate. 2 μL of active β-secretase (protein concentration: 4 μg/μL) was added to the 50 μL of extraction buffer as the positive control. For negative control, 2 μL of the β-secretase inhibitor was added to the 50 μL of sample well. 2 μL of each of the tested samples were added into each sample well for inhibitory activity evaluation. Following compounds addition, 50 μL of 2X reaction buffer was added with a gentle mix and incubation for 20 min at 37 °C before adding 2 μL of the β-secretase substrate. The plate was then covered and incubated in the dark at 37 °C for 1 h. Samples were then measured in a fluorescent 96-well plate reader (Ex/Em = 345/500 nm). Background readings produced from the substrate without adding secretase were subtracted from all samples.