Protein a donor beads

Notch1 substrate (N1-Sb1) (0.4 μM) was incubated with cell membrane from the cell pellet (40 μg/mL) in the presence of 0.25% CHAPSO and PIPES buffer (50 mM PIPES at pH 7.0, 150 mM KCl, 5 mM CaCl₂ and 5 mM MgCl₂) in a sealed 384-well plate at 37 °C for 2 h. Twenty microliters of the reaction mixture were transferred into a clean well. Twenty microliters of the AlphaLISA assay mixture was transferred into the same well. The AlphaLISA mixture consists of 0.2 μg/mL of SM320 antibody, protein A donor beads (5 μg/mL) (PerkinElmer Life Sciences, USA) and Streptavidin acceptor beads (20 μg/mL) (PerkinElmer Life Scicences, USA). The plate was then sealed and shaken briefly to mix the reaction followed by overnight incubation. The signal was detected using Envision plate reader at 615 nm (PerkinElmer Life Sciences, USA). The signals generated were an arbitrary unit of gamma-secretase activity.