Characterization of APP complexes by native-PAGE and sucrose gradient ultracentrifugation. (A) APP complexes from the brain (frontal cortex from NDC subjects) and CSF samples (NDC subjects) were analyzed by blue native-PAGE. Incubation of blots with antibodies for the different APP epitopes confirmed the presence of APP dimers in brain extracts and CSF samples (~242 kDa), but also the existence of APP complexes with higher molecular weight. A CSF sample denatured by boiling at 95°C for 5 min under fully reducing conditions (Dn) was also analyzed by blue native-PAGE to warrant the migration of the monomeric sAPP band. (B) Brain extracts and CSF samples were also fractionated on 5-20% sucrose density gradients. The fractions (collected from the top of each tube) were immunoblotted for APP with the 6E10 antibody, and additionally with IBL-β and Sigma-Ct antibodies for CSF samples. Enzymes of known sedimentation coefficient, β-galactosidase (G, 16.0S; ~540 kDa), catalase (C, 11.4S; ~232 kDa) and alkaline phosphatase (P, 6.1S; ~140-160 kDa) were used as internal markers.