Preparation and characterization of Aβ monomers and Aβ oligomers. A. Preparation of Aβ monomers and Aβ oligomers. Synthetic Aβ peptides were aged for various amounts of time in phosphate buffer followed by SEC fractionation, yielding two major peaks corresponding to Aβ oligomers (AβO) and Aβ monomers (AβM), with Aβ oligomers eluting at 12–15 min and Aβ monomers eluting at 25–30 min. With increased aging, the relative amount of Aβ oligomers increased to a maximum at 24 h. The Z-axis is the relative absorption intensity at 220 nm. B. Western blot analysis of SEC fractionated in vitro preparations of Aβ monomers (AβM) and Aβ oligomers (AβO) with the Aβ specific antibody 6E10, revealed a single, 4.5 kDa monomeric band for AβM and monomer, dimers, trimers, tetramers and large molecular weight oligomers that run between 28–90 kDa (high 'n' AβOs) for AβO. C, D. Western blot analysis of tissue culture media with 0.2 μg/ml (lanes 1 and 6), 0.4 μg/ml (lanes 2 and 7), 0.8 μg/ml (lanes 3 and 8), 2.0 μg/ml (lanes 4 and 9) and 4.0 μg/ml (lanes 5 and 10) of AβM and AβO at the beginning (time = 0 h, C) or end (time = 24 h, D) of the experiment revealed a similar migration pattern of AβM and AβO at both time points. Asterisk indicates the location of abundant proteins in the B27 serum-free supplement.