Lipid Vesicle Preparation for α-Synuclein

Wild type α-syn was expressed and purified as previously described 36 (link),51 (link). The lipids were dissolved in 20 mM phosphate buffer (NaH₂PO₄/NaH₂PO₄), pH 6.5, and stirred at 45°C for two hours. The solution was then frozen and thawed 5 times using dry ice and a water bath at 45°C, respectively. The preparation of small or large unilamellar vesicles, SUVs or LUVs, respectively, was done using sonication (3 × 5 min, 50 % cycles, 10 % maximum power) on ice or extrusion through 100 nm pore diameter membranes (Avanti Polar Lipids, Inc) at 45°C, respectively. After centrifugation, the sizes of the SUVs and LUVs were checked using dynamic light scattering (Zetasizer Nano ZSP, Malvern Instruments, Malvern, UK) and show to consist of a distribution centred at 20 and 100 nm diameter, respectively.