SDS-PAGE, immunoblotting and Coomassie staining

Samples were loaded onto a 12% Tris-Glycine SDS-PAGE gel. PageRuler Plus Prestained Protein Ladder (Thermo Fisher Scientific) was used as a molecular weight marker. The gel electrophoresis was performed at 90 V for approximately 30 min, then at a constant voltage of 120 V until the dye front ran off the end of the gel. Gels were used for immunoblotting or Coomassie staining.

Proteins were transferred onto nitrocellulose membranes (GE Healthcare) for 120 min at 250 mA by Criterion Blotter (Bio-Rad). The membranes were blocked with 5% non-fat milk in TBS-T (200 mM Tris, 1.5 mM NaCl, and 0.05% Tween-20) for 1 h, after which they were incubated overnight at 4°C with anti-PrP SAF 84 antibody diluted 1:500 in the blocking solution. Membranes were washed three times with TBS-T, after which they were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody goat anti-mouse diluted 1:1000 in blocking solution. After three washes again with TBS-T, the signal was detected using enhanced chemiluminescence (GE Healthcare), and the band intensity was acquired using the UVI Soft software (Uvitec Alliance, Cambridge).

Gels were also stained in GelCode Blue Safe Protein Stain (Thermo Fisher Scientific) by gently shaking for 1 h. Gels were destained overnight in ultrapure water and used further for in-gel trypsin digestion.