Cell membrane was used as the source of γ-secretase and was prepared as previously described (15 (link),16 (link)). The in vitro γ-secretase activity assay was performed according to previously reported literatures (17 (link),18 (link)). Briefly, recombinant APP substrate CT6 (1uM) was incubated with membrane protein (40 μg/ml) and 0.25% CHAPSO for 3 hours at 37 °C. To measure enzyme inhibition for Aβ cleavage, the activity assay was carried out in the absence or presence of various concentrations of inhibitors. The reaction (20ul) was then combined 1:1 with detection mix (20ul), which includes streptavidin-conjugated Alpha donor beads (PerkinElmer Life Sciences), anti-mouse IgG AlphaLISA acceptor beads (PerkinElmer Life Sciences) and Aβ40 specific antibody G2–10. The sample was incubated in the dark at room temperature overnight, and the AlphaLISA signal was detected with an Envision plate reader (PerkinElmer Life Sciences).
Protocol detail
In Vitro Assay of γ-Secretase Activity
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Anti igg
Antibody
Assay
Cell membrane
Chapso
Cleavage
Donor
Enzyme
Inhibition
Inhibitors
Membrane protein
Mouse
Secretase
Streptavidin
Corresponding Organization : Cornell University
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Variable analysis
independent variables
- Various concentrations of inhibitors
- Enzyme inhibition for Aβ cleavage
- Recombinant APP substrate CT6 (1uM)
- Membrane protein (40 μg/ml)
- 0.25% CHAPSO
- Incubation for 3 hours at 37 °C
- Positive control: Assay in the absence of inhibitors
- Negative control: Not explicitly mentioned
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