Figure 2. Urea gels show Aβ 1-x products in different phases of γ-secretase reaction.

The reactions were prepared using CHAPSO enriched γ-secretase membranes (total protein 0.25 mg/ml), and saturating concentration of C99 substrate (3.0 µM). The lanes “Aβ std 1-x” represents synthetic peptides as mobility standards. To facilitate detection of the early data points ( A ) the reaction volume was twenty fold bigger than usual, and the resulting 1-x Aβ products were concentrated about twenty-fold by immunoprecipitation using protein G beads and polyclonal antibodies specific for the first 5 amino acids. It is necessary to mention that pre-incubation of the assay mix for several hours prior to the start of reaction (i.e. addition of C99 substrate) does not affect the relative distribution of different Aβ products. Therefore, the observed changes are not due to enzyme denaturation during the course of the reaction.