Western Blot Detection of Prion Proteins

Methods were performed as described previously [26 (link)]. Briefly, 20 μl of each amplified product was incubated at 45°C with PK (200 μg/ml) for 1 hr before denaturation at 100°C in SDS-PAGE sample buffer. Samples were run on 12% NUPAGE gels and electrotransferred onto PVDF membrane. Western blot (using the SNAP ID system, Millipore) was performed using 3F4 or 6D11 anti-PrP monoclonal antibodies for human and sheep prion detection, respectively (Eurogentec, 49001 Angers, France), and anti-mouse IgG peroxidase-linked secondary antibody (GE Healthcare, UK) linked to a chemiluminescent reaction (ECL blotting detection reagent, GE Healthcare life sciences, Amersham, France).