The expression and purification procedure for the peptide Aβ(M1-42) (MDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA) and the molecular chaperones clusterin and proSP-C Brichos were carried out as described in previous papers (4447). In short, Aβ(M1-42) inclusion bodies were extracted from Escherichia coli cells by sonication and dissolved in 8 M urea. Further purification was performed by ion exchange in batch mode on DEAE cellulose resin with additional lyophilization and gel filtration on a 3.4 cm × 200 cm gel-filtration column at 4°C (44).

To obtain the proSP-C Brichos domain, E. coli cells were lysed by lysozyme (1 mg/ml) for 30 min and incubated with deoxyribonuclease and 2 mM MgCl2 for another 30 min on ice. The centrifuged cell pellet was dissolved in 2 M urea in 20 mM tris and 0.1 M NaCl (pH 8) and sonicated for another 5 min. After another centrifugation step, the supernatant was filtered through a 5-μm filter and purified with a 2.5-ml nickel-agarose column. The thioredoxin and His6 tag were removed by adding thrombin for 16 hours at 4°C, followed by another run through a nickel column. The protein was further purified by ion exchange chromatography (45, 46).

Clusterin was extracted from human blood plasma obtained from Wollongong Hospital (Wollongong, New South Wales, Australia). Complete protease inhibitor was added, and the mixture was filtered through (i) a GF/C glass fiber filter and then (ii) a 0.45-mm cellulose nitrate filter. The filtrate was further purified on a 5-ml G7 anti-CLU monoclonal antibody column. After severe washing steps, the specifically bound material was eluted using 2 M GdnHCl in phosphate-buffered saline (PBS). The fraction was dialyzed against 20 mM MES (pH 6.0) and loaded on a 1-ml HiTrap SP XL cation exchange column, collecting the flow through. Last, the pure protein was obtained by size exclusion chromatography on a Superose 6 10/300 column (47). For the microfluidic experiments, clusterin was covalently labeled with Alexa Fluor 488 N-hydroxysuccinimide ester (Thermo Fisher Scientific, Waltham, USA). To achieve this, the protein (2 mg/ml) was incubated with a 10-fold molar excess of the fluorophore (added from a 10 mM stock in dimethyl sulfoxide) [1 hour, room temperature (RT)]. Unconjugated dye was removed by buffer exchange into PBS using a PD-10 column (GE Healthcare, Chicago, USA). The final protein concentration and labeling efficiency was measured according to the manufacturer’s instructions. We carried out kinetic experiments to show that the labeling with the Alexa dye, required for the fluorescence detection, does not modify the inhibition and the binding properties of clusterin (fig. S6). BSA, obtained from Sigma-Aldrich (St. Louis, USA), was used. All aggregation assays and binding reactions were carried out in 20 mM sodium phosphate buffer at pH 8.0. All chemicals were of analytical grade and purchased from Sigma-Aldrich, unless otherwise stated.

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