The human Aβ1-42 peptide, HFIP-pretreated (Anaspec) was diluted (5 mM) in anhydrous DMSO and then in phenol red-free Ham’s F12 medium (PanBiotech) at 100 μM as previously described68 (link). Aliquots were immediately frozen and stored at −80 °C. Before aggregation, 100 μM Aβ1-42 in Ham’s F12 was diluted at 65 μM by adding scFvA13 or NaPB buffer (NaCl 100 mM, sodium phosphate buffer pH7, 20 Mm) as control. The scFvA13 was used at stoichiometric (65 μM) or substoichiometric (13 μM) ratio with Aβ1-42. The aggregation was performed at 22 °C for 24 h. For disassembly assays, stoichiometric (65 μM) or substoichiometric (13 μM) concentrations of the scFvA13 or the NaPB buffer as control were added to Aβ1-42 in Ham’s F12 (preassembled at the concentration of 100 μM, 22 °C for 24 h) obtaining a final concentration of 65 μM Aβ. The disassembly assay was performed at 22 °C for 24 h.
Synthetic Aβ samples, not boiled nor treated with reducing agents, were loaded (30 ng per well) in Bis-Tris XT Criterion Gels (Bio-Rad), run in MES buffer and analysed by WB, as described above by 6E10 (Covance), 4G8 (Covance) or by a rabbit mAb anti-Aβ (D54D2, Cell Signalling).
Synthetic Aβ samples, not boiled nor treated with reducing agents, were loaded (30 ng per well) in Bis-Tris XT Criterion Gels (Bio-Rad), run in MES buffer and analysed by WB, as described above by 6E10 (Covance), 4G8 (Covance) or by a rabbit mAb anti-Aβ (D54D2, Cell Signalling).