Western Blot Analysis of PrP Isoforms

Samples were loaded onto 12% Tris-Glycine SDS-PAGE and transferred onto Immobilon P PVDF membranes (Millipore, Burlington, MA, USA) for 2 h at 4 °C. Membranes were blocked in 5% non-fat milk in TBST (Tris 200 mM, NaCl 1.5 mM, 1% Tween-20, Sigma-Aldrich) for 1 h at RT. The following primary antibodies were incubated overnight at 4 °C: anti-PrP W226 1:1000 [41 (link)] and anti-PrP EB8 1:1000 [43 (link)]. After three washes in TBST, membranes were incubated with goat anti-mouse IgG conjugated with horse-radish peroxidase for 1 h at RT and developed using Immobilon Classico Western HRP substrate (Millipore). Anti-β-actin antibody 1:10,000 (Sigma-Aldrich) was incubated for 1 h at RT and used for normalization. Images were acquired with Uvitec Alliance (Cambridge, UK) and Uviband analysis software was used for densitometry analysis.