Immunoblotting Protocol for Alpha-Synuclein

For immunoblotting, samples were dissolved in loading buffer (4% SDS, 40% glycerol, 1% bromophenol blue, and 50 mM Tris, pH 6.8 supplemented with 0.5 M dithioerythritol), denatured at 95°C for 5 min before being resolved on 10% to 16% polyacrylamide gels and transferred onto polyvinylidine difluoride (PVDF) membranes (GE Healthcare, UK). To enhance visualization of α-syn, the membranes were fixed in 4% paraformaldehyde (PFA) in PBS for 30 min followed by boiling in PBS for 5 min as previously described (81 (link)). PVDF membranes were blocked in 5% nonfat milk dissolved in TBST (10 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween-20) for 1 h at RT, incubated with primary antibodies overnight at 4°C, washed, and incubated with horseradish peroxidase (HRP) conjugated secondary antibodies (DAKO, Denmark) for 1 h at RT. Proteins were visualized with enhanced chemiluminescence using a Fuji LAS-3000 Intelligent Dark Box (Fujifilm, Japan).