2.5. Proteinase K Digestion and Western Blotting

Proteinase K (PK) digestion of the reaction end products was performed using two different proteinase K concentrations (0.4 and 4 µg/mL) at 37 °C for 30 min. The reactions were stopped by adding NuPAGE LDS buffer and incubating the samples at 60 °C for 10 min. Samples were analyzed by electrophoresis in 4%–12% Bis-Tris gels (Invitrogen) using MES as running buffer and immunoblotted on nitrocellulose membranes (Amersham). Blots were blocked in PBS containing 0.05% (v/v) Tween 20 (PBST) and 5% (w/v) non-fat dried skimmed milk powder and probed with Syn1 antibody (aa91-99 of human αSyn) at 50 ng/mL final antibody concentration in PBST. Blots were developed using ECL detection Western blotting reagents (Pierce).