Hydrobond c extra

For Western blotting, samples were boiled for 5 min at 95°C for in loading buffer (50 mM Tris pH 6.8, 40% glycerol, 4% SDS, bromophenol blue) without reducing agent before loading onto a 4–16% PAGE gel (GenScript ExpressPlus™). Gels were blotted on PVDF membranes and fixed in 4% formaldehyde for 30 minutes and boiled in PBS for 5 minutes. For dot blotting 50-100ng of protein was blotted on nitrocellulose (Hydrobond-C Extra, GE Healthcare) and these filters were not fixed or boiled. All membranes were blocked in non-fat milk (TBS with 5% non-fat milk, 0.05% Tween 20 and 0.02% sodium azide) for 30 minutes at RT followed by incubation with primary antibodies overnight. membranes were washed in TBS-T (TBS with 0.1% Tween) and incubated with secondary antibodies for 1.5 hour. Membranes were washed, developed using ECL reagent (Pierce™, Thermo Scientific), and subsequently imaged on a LAS-3000 imaging system (Fuji). Blot quantification was done using ImageJ

Dot blotting was performed on 100 ng of protein blotted on nitrocellulose (Hydrobond-C Extra, GE Healthcare). Prior to loading, α-syn was incubated for 30 min at a concentration of 10 µg/mL (0.69 µM) in PBS alone or in combination with 0,1%, 0,5%, 1%, 2%, 5% and 10% of DMSO at room temperature (RT). All membranes were blocked in non-fat milk (Tris-buffered saline (TBS) with 5% non-fat milk, 0.05% Tween 20 and 0.02% sodium azide) for 30 min at RT followed by incubation with primary antibodies overnight. Membranes were washed in TBS-T (TBS with 0.1% Tween) and incubated with secondary antibodies for 1.5 h. Membranes were washed, developed using ECL reagent (Pierce™, Thermo Scientific), and subsequently imaged on a LAS-3000 imaging system (Fuji). Blot quantification was done using ImageJ.

Dot blotting was performed as previously described (83 (link)) where 100 ng of protein was blotted on a nitrocellulose membrane (Hydrobond-C Extra, GE Healthcare). All membranes were blocked in nonfat milk (TBS with 5% nonfat milk, 0.05% Tween 20 and 0.02% sodium azide) for 30 min at RT followed by incubation with primary antibodies overnight. Membranes were washed in TBS-T (TBS with 0.1% Tween) and incubated with secondary antibodies for 1.5 h. Membranes were washed, developed using ECL reagent (Pierce, Thermo Scientific), and subsequently imaged on a LAS-3000 imaging system (Fuji).