Adhesive plate sealers

One milligram lyophilisates were dispersed in denaturant 1 (7 M GuHCl and 5 mM EDTA), incubated overnight at room temperature, and monomer isolated by size exclusion chromatography (SEC) on a Superdex 75 10/300 column eluted with Aβ buffer (20 mM NaPO₄, 0.2 mM EDTA, 0.02% NaN₃, pH 8.0). Only the peak fractions were retained for aggregation assays. Aggregation was monitored using a continuous thioflavin-T (ThT) fluorescence assay performed essentially as described [38 (link)]. SEC-isolated peptide was diluted to 20.1 μM with Aβ buffer and combined with ThT to yield 30 μM ThT and 20 μM Aβ. Six replicates (100 μl) were transferred to wells of black, clear bottom, half-area PEG-ylated 96-well plates (#3881; Corning Life Sciences, Corning, NY). Reactions prepared in the absence of ThT were used for downstream fibril harvest, and blanks without any Aβ were included. Plates were covered with adhesive plate sealers (VWR, Radnor, PA) and incubated at 37 °C in a POLARstar Omega plate reader (BMG Labtech, Ortenberg, Germany). Fluorescence was recorded every 5 min (Ex₄₄₀ and Em₄₈₀) and reactions were carried out for at least 15 h.

Three-milligram lyophilisates were dispersed in denaturant 2 (6 M GuHCl, 25 mM Tris and 5 mM EDTA), incubated for 2 h at room temperature and chromatographed on a Superdex 75 10/300 column eluted with αSyn buffer (10 mM MES, 140 mM NaCl, pH 5.5). As with Aβ, only the peak fractions were retained for aggregation assays. Aggregation was monitored using a continuous ThT assay [35 (link)], with modification. SEC-isolated peptide was diluted to 40 μM with αSyn buffer and combined with ThT to yield 20 μM ThT and 35 μM αSyn. Six replicates (100 μL) were transferred to wells of black, clear bottom, full-area nontreated 96-well plates (#3631; Corning Life Sciences, Corning, NY). Reactions prepared in the absence of ThT were used for downstream fibril harvest, and blanks without any αSyn were included. Plates were covered with adhesive plate sealers (VWR, Radnor, PA) and incubated at 37 °C in a POLARstar Omega plate reader (BMG Labtech, Ortenberg, Germany) with shaking at 100 RPM for 300 s before each reading. Fluorescence was recorded every 10 min (Ex₄₄₀ and Em₄₈₀) and initial reactions were carried out for at least 96 h.