Orbital mixer

Full-length, wildtype unmodified α-Syn was incubated at 37°C for one week under continuous shaking in an Eppendorf Thermomixer set at 600 r.p.m., to assemble into fibrillar form. 700 μM α-Syn was assembled in 50 mM Tris-HCl, pH 7.5, 150 mM KCl buffer (Table 1).
To prepare fibrils of full-length, phosphorylated, N-terminally acetylated, N-terminally acetylated and the E46K mutant α-Syn, recombinant protein (dialyzed and lyophilized) was diluted to 5 mg/mL in 200 µL of Dulbecco’s phosphate buffered saline (DPBS) buffer (Gibco; 2.66 mM KCL, 1.47 mM KH₂PO₄, 137.93 mM NaCl, 8.06 mM Na₂HPO₄-7H₂O pH 7.0–7.3). After 5 days of incubation at 37°C with constant agitation (1,000 rpm) in an orbital mixer (Eppendorf), reactions were sonicated for 5 min in a Branson 2510 water bath, aliquoted, and stored at −80°C. All fibrils were created in the presence of an air-water interface. The presence of amyloid fibrils was confirmed by thioflavin T fluorimetry and high molecular weight assemblies were visualized by gel electrophoresis.

The mouse recombinant full-length α-Syn monomer was purified by following the instructions^{24 (link)}. Briefly, after serial purification steps using Superdex 200 Increase 10/300 G size-exclusion and Hitrap Q Sepharose Fast Flow anion-exchange columns (Cytiva), the α-Syn monomers were subjected to HiTrap SP HP cation-exchange column (Cytiva) for removing lipopolysaccharides. The α-Syn fibrils were mixed together by stirring in an Eppendorf orbital mixer for 7 days at 1,000 rpm and 37°C. Before use, the α-Syn monomer was kept at 80°C. α-Syn fibrils were sonicated for 30 sec (0.5 sec pulse on/off) using a Branson Digital sonifier with a 10% amplitude.