Figure 5. Oligomerization of C99 substrate.

C99 dimerization/oligomerization was measured using aliquots of C99 substrate that had high activity with γ-secretase in CHAPSO enriched membranes. Oligomerization between C99 molecules was measured using AlphaScreen® technology by coupling both the donor-beads, and the acceptor-beads, to 3D6 antibody (right panel). Increasing concentration of C99 substrate was incubated with 10 nM of 3D6 monoclonal antibodies coupled to either donor or acceptor-beads. Since one epitope can bind only one antibody, the acceptor and the donor beads can come to proximity and give the AlphaScreen® signal only if C99 dimerization/oligomerization brings the epitopes together (right panel). A nonlinear regression and the equation 5 (methods) were used to calculate an apparent dissociation constant, Kd = 33±2 nM [69].