Immunoblot detection of PrPSc.

Brain homogenates (10%, wt/vol) were prepared in NP-40 lysis buffer (1% NP-40, 0.5% sodium deoxycholate, 150 mM NaCl, 50 mM Tris HCl [pH 7.5]) and incubated at 37°C for 1 h with 20 μg/ml PK. Digestions were halted by the addition of 1 mM phenylmethylsulfonyl fluoride. Samples were then subjected to electrophoresis through 12% Tris-glycine polyacrylamide gels (NuPAGE; Life Technologies) and transferred to polyvinylidene difluoride (PVDF) membranes by semidry blotting. PrP was detected using anti-mouse PrP-specific monoclonal antibody (mAb) 7A12 (69), followed by horseradish peroxidase-conjugated goat anti-mouse antibody (Jackson ImmunoResearch), and visualized by chemiluminescence (BM chemiluminescent substrate kit; Roche, Burgess Hill, UK).