Thioflavin T (ThT)-based aggregation assay

Peptide (pWT and pR248W) amyloid formation kinetics were measured in quadruplicate in black 96-well plates with flat bottom (Corning Inc., NY) using a Synergy H1MF Multi-Mode microplate reader controlled by Gen5 software (version 2.0; BioTek, Winooski, VT). The aggregation was initiated by dilution of peptide from a freshly prepared stock solution (1 mM in DMSO) to PBS containing ThT, with or without oligopyridylamides. For some experiments, the oligopyridylamides were added (from a stock solution of 10 mM in DMSO) at the indicated timepoint after the start of the aggregation reaction. To maintain identical conditions, an equal amount of DMSO was added to the wells with peptide only reactions. Final concentrations in the wells were: 25 μM pWT or pR248W; 0 or 25 μM oligopyridylamide; 50 μM ThT. Peptide aggregation was monitored by shaking and measuring the ThT fluorescence (λ_ex/em = 440/480 nm) at 5-min intervals at 37 °C. The sample data were processed by subtracting the blank and renormalizing the fluorescence intensity by setting the maximum value to one.