Preparation and Characterization of Amyloid-Beta Monomers

Synthetic peptide Aβ(1-42): [H2N]-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA-[COOH] was purchased from Biopeptide. Preparation of the monomeric form of Aβ(1-42) was performed as described elsewhere37 (link). To do this, cold hexafluoroisopropanol (Fluka) was added to dry Aβ (1-42) to a concentration of 1 mM and incubated for 60 min at room temperature. Then this solution was put on ice for 10 min and aliquoted into non-siliconized microcentrifuge tubes (0.56 mg peptide per tube). Peptide in the tubes was dried under vacuum using Eppendorf Concentrator 5301. Dried peptide was stored at −80 °C. 2.5 mM peptide stock solution was prepared by adding 20 μL of 100% anhydrous DMSO (Sigma-Aldrich) to 0.22 mg peptide and incubating for 1 h at room temperature. For use in the experiments, the peptide was diluted to the required concentration with buffer solution. Equivalent amount of DMSO was added to the control samples in all experiments. Only freshly prepared peptide solutions were used for all experiments. By dynamic light scattering (DLS) and turbidity methods it was shown that there were no aggregates and higher molecular weight oligomers in Aβ(1-42) preparation (40 μM) after 1 hour following preparation of the water solution. To determine the turbidity the optical density at 405 nm of freshly prepared solutions of Aβ(1-42) was recorded on Jasco V-560 spectrophotometer within one hour. The absorbance of the solution does not change, which allows to make a conclusion about the absence of particles in solution with a size of 1–100 nm. DLS method allows measuring the particle size from 0.6 to 10 nm. The DLS measurements were carried out on a Zetasizer Nano ZS apparatus (Malvern Instruments Ltd.). According to our data the freshly prepared solution of amyloid (40 μM) does not contain particles in this size range.
The monomer and low molecular weight oligomer quantities in Aβ(1-42) solution were estimated by SDS-PAGE with pre-stabilization of oligomers by photoinduced crosslinking using covalent Tris (2,2-bipyridyl) dichlororuthenium (II) hexahydrate38 (link). The monomers constituted 80% in Aβ(1-42) preparation (Supplementary Figure S8).
Purified preparation of Na,K-ATPase (α1β1 isozyme) was obtained from duck salt glands as described elsewhere11 (link)39 (link). The purity grade of Na,K-ATPase was 99% (Supplementary Figure S9) and specific activity of the enzyme reached ~2400 μmol of Pi (mg of protein × h)⁻¹ at 37 °C.