Fig. 4: ThT-based aggregation assays reveal different aggregation behaviour for aSyn familial mutants.

Aggregation kinetics of aSyn WT and familial mutants A30P, A53T, E46K, A53E, H50Q, G51D were measured using ThT fluorescence intensity and plotted as % of maximum fluorescence a in the absence of calcium and b in the presence of 2.5 mM CaCl₂ (plate repeat data are available in Supplementary Fig. 12). 20 μM aSyn was incubated with 20 μM ThT in a half area 96 well plate with orbital agitation at 300 rpm for 5 min before each read every hour for 150 h. c Lag time (t_lag) and d time to reach 50% of maximum aggregation (t₅₀) were calculated and the mean is numerically shown (t_lag: *p = 0.0145, **p = 0.0034, ***p = 0.001, t_50: **p = 0.001, ***p = 0.0003, ***p = 0.0004, *p = 0.043, *p = 0.002)). Mutant G51D did not reach the elongation phase in the studied timeframe, so the t₅₀ value was not calculated in (c). e The remaining monomer concentration was determined using SEC-HPLC, 35 μL of monomer from each well in the ThT assays was analysed on an AdvanceBio SEC 130 Å column in 20 mM Tris pH 7.2 at 1 mL min⁻¹. Remaining monomer concentrations were measured from the area under the peak and calculated using a standard curve of known concentrations. The mean remaining monomer concentration is numerically shown (*p = 0.0438, *p = 0.0466). Measurements were repeated using at least four sample replicates in three experiments and an unpaired two-tailed t-test with a 95% confidence interval was used to calculate statistical significance between samples before and after the addition of calcium. Error bars represent s.d.