Figure 1.
Density gradient ultracentrifugation.
Schematic picture of the density gradient ultracentrifugation setup and the fractionation into four fractions, with the largest structures in the first fraction and the smallest structures in the fourth fraction.
Figure 2.
Fractionation of synthetic Aβ1–40 and Aβ1–42.
Each of the four fractions of the Aβ40 and Aβ42 preparations was analyzed with ELISA for total levels of Aβ1–40 and Aβ1–42 respectively as well as for protofibril levels (A–B). Graphs show mean of three different fractionated Aβ preparations and error bars indicate the standard deviation. Equal amounts of Aβ (0.1 µM) from the Aβ1–42 fractions were also analyzed with MTT cell viability assay with PC12 cells (C). The MTT graph shows the mean of three analyses of three different fractionated Aβ1–42 preparations. The analyses are standardized with an internal Aβ control and all fractions were diluted to a final Aβ concentration of 0.1 µM and an optiprep concentration of 1.5% prior to addition to the cells. Error bars indicate the standard deviation. Aβ1–42 fractions were also analyzed with AFM (D–G). Scale bar = 100 nm. Curves under AFM images represent the topography of the area marked by a line in each image. An AFM image of a non centrifuged preparation of Aβ1–42 is shown for reference (H). Arrows point at structures resembling those present in the four different fractions of centrifuged Aβ1–42.
Table 1.
Molecular sizes of fractionated synthetic Aβ42, determined with AFM.
Figure 3.
Fractionation of transgenic mouse brain extracts.
Fractions of mouse brain homogenate from AβPPArcSwe transgenic mice (n = 5) and non-transgenic littermates (n = 5) were analyzed with Aβ1–42 (A), Aβ1–40 (B) and Aβ protofibril specific (C) ELISAs. Horizontal lines indicate the mean value of each group. Immunoprecipitation of pooled material from fraction 2 of transgenic mouse brain homogenate with the conformation specific Aβ protofibril selective antibody mAb158 covalently coupled to Dynabeads (0 – non immunoprecipitated sample, IP – immunoprecipitated material, S – supernatant remaining after ip) (D). Error bars indicate the standard deviation.
Figure 4.
Fractionation of human brain extracts.
Fractions of human brain homogenate from temporal cortex of diseased AD patients (n = 7, including one AβPPSwe and one AβPPArc mutation carrier) and non-AD subjects (n = 4, one control subject and three FTD patients) were analyzed with Aβx-42 (A), Aβx-40 (B), Aβ1–42 (C) and Aβ1–40 (D) ELISAs. Results from the individual carrying the Swedish mutation is marked ‘Swe’ in the Aβx-40 and Aβ1–40 graphs. Horizontal lines indicate the mean value of each group. The level of N-terminal truncation of Aβ42 was determined as a ratio between Aβ1–42 and Aβx-42 (1-[Aβ1–42]/[Aβx-42]) (E), with error bars indicating the standard deviation. Immunoprecipitation of pooled material from fraction 2 of AD brain and non-AD brain with the conformation specific Aβ protofibril selective antibody mAb158 covalently coupled to Dynabeads (0 – non immonuprecipitated sample, IP – immunoprecipitated material, S – supernatant remaining after ip) (F). Error bars indicate the standard deviation.
Table 2.
Summary of cases.