Western Blot Analysis of Brain PrPres

Brain PrP^res was analysed as previously described^{7 (link),14 (link)}. Briefly, 175 ± 20 mg of frozen brain tissue were homogenized in 5% glucose in distilled water in grinding tubes (Bio-Rad) adjusted to 10% (w/v) using a TeSeE^TM Precess 48^TM homogenizer (Bio-Rad) following manufacturer instructions. Presence of PrP^res in transgenic mice brains was determined by Western blot, following the procedure described below and using the reagents of the ELISA commercial test (TeSeE, Bio-Rad). Based on a previously described protocol^{48 (link)} 10–100 μl of a 10% (w/v) brain homogenate were treated with proteinase K and the resulting samples were loaded in 12% Bis-Tris Gel (Criterion XT, BioRad). Proteins were transferred electrophoretically onto PVDF membranes (Millipore). For immunoblotting, monoclonal antibodies 12B2 and Sha31^{49 (link)} were used at a concentration of 1 µg/mL. Sha31 recognizes ₁₄₅-YEDRYYRE-₁₅₂ epitope of the macaque/human PrP^C sequence. Immunocomplexes were detected as described above for brain PrP^C analysis. N-glycosidase F (PNGaseF, New England Biolabs) was used according to manufacturer’s instructions with minor modifications.