Aggregation of human recombinant alpha-synuclein

Humanrecombinant α-Syn Monomeric WTα-Syn is purified from Escherichia coli as

previously described in

[Hoyer et al., 2002].

Aggregation reactions are carried out using a solution of α-Syn 70micromolar (µM) in 25millimolar (mM) Tris buffervsupplemented with 100millimolar (mM) NaCl, 7.4 (in the presence of 0.01% volume % NaN3 to prevent bacterial growth).

The buffer is freshly prepared before each experiment and passed through a 0.02 µm

syringe filter (Anotop, Whatman) to remove insoluble contaminants.

Prior to incubation, the reaction mixture is ultra-centrifuged at 90k r.p.m. for 1h 0m 0s at 4°C to remove potential seeds.

The supernatant is collected and separated in two fractions: one kept at 4°C at all

times until use(monomers), and a second incubated in the dark at 37°C37°C and200rpm,0h 0m 0s , during7h 0m 0s -8h 0m 0s hours to avoid fibril formation (monomers+oligomers).

α-Syn is always kept in LoBind microcentrifuge tubes (Eppendorf, Hamburg, Germany) to

limit surface adsorption.