Generation and characterization of oligomer-specific antibody. (A) Electrophoresis of immunogen. SDS-PAGE was performed to isolate the Aβ1-42 tetramer (red closed arrowhead) alone without any contamination by the Aβ1-42 trimer (black closed arrowhead) and Aβ1-42 monomer (opened arrowhead). Lane 1, Aβ1-42 dissolved in 10 mM phosphate buffer; lane 2, Aβ1-42 dissolved in distilled deionized water. (B) Aβ1-42 oligomer formation was observed as a function of time. Aβ1-42 monomer (25 μM) incubated at 37°C for the indicated time (0 - 72 h) were spotted on nitrocellulose membrane and subjected to a dot blot assay using A11 (1:100), 1A9 (1:50), 2C3 (1:50), or 4G8 (1:1000). (C) In this dot blot assay (left half of panel C), 1 μg of soluble Aβ42 oligomers (100,000 g sup for 4-h-incubation at 37°C) and Aβ42 fibrils (100,000 g pellets for 120-h-incubation at RT) were applied on a nitrocellulose membrane and probed with A11, 1A9, 2C3, or 4G8. EM image of fibrils (right half of panel C). (D) Characterization of Aβ1-42 oligomers under nondenaturing conditions. Aβ1-42 monomer (25 μM) incubated at 37°C for 4 h was separated on 16% BN-PAGE. (E) Separated peptides under nondenaturing conditions were also subjected to immunoblot analysis using A11, 1A9, 2C3, and 4G8. (F) The 100000 g sup of 4-h-incubated mixture of Aβ1-42 monomer (25 μM) was subjected to two-dimensional native/SDS-PAGE, followed by 4G8-immunoblot analysis. SDS-stable 15~40-mers are indicated (] (red)). (G) Immunodetection of 4-h-incubated mixture of Aβ1-42 monomer (25 μM) under denaturing conditions probed with A11, 1A9, 2C3, and 4G8. SDS-stable 15~40-mers are indicated (] red).