PrPs were separated by SDS-PAGE, and the gels immersed in 20% methanol/5% acetic acid. The gels were then observed under UV light (365 nm) and stained with Coomassie brilliant blue. A cell-free conversion reaction assay was carried out as described previously (12–14). Seed PrPScs (600 ng) were pretreated with 3

M Gdn-HCl at 37°C for 1 hr. Recombinant PrPs (10 pmol) were added to conversion buffer (1.5 M Gdn-HCl, 1.25% sarkosyl, 50 mM citrate, pH 6.0). Pretreated seed PrPSc was then added to a final reaction volume of 24 μL, and the mixture incubated at 37°C for 24 hr. After incubation, PK (20 μg/mL) digestion was carried out at 37°C for 30 min. The digestion was stopped by adding pefabloc (Roche, Indianapolis, IN, USA) to a final concentration of 2 mM. Proteins in the reaction mixture selleck were recovered by precipitation with four volumes of methanol, and the pellets were dried and dissolved in SDS-PAGE sample buffer containing 100 mM DTT. The recovered de novo PrPress were analyzed by Western blotting using 3F4 mAb, and the signal intensities evaluated using a Chemi imager (Alpha Innotech) . The conversion efficiencies check details for each sample were expressed as a percentage change (mean ± SE relative to a control

value [control = 100]). The Chandler-seeded conversion value of MoPrP under nonreducing conditions was used as a control. A binding assay was performed as described previously (14). After the same incubation as in the cell-free conversion assay, the reaction mixture was centrifuged at 20,000 g for 10 min at 25°C. The supernatant (unbound fraction) was transferred to a tube, and the pellet (bound fraction)

was resuspended in 24 μL of the reaction solution, followed by washing once with 100 μL of the reaction solution. All samples were methanol-precipitated, and SDS-PAGE and Western blotting were conducted as described above. Binding efficiencies were expressed as a percentage change (mean ± SE) ADP ribosylation factor relative to control values. The binding value of MoPrP with Chandler PrPSc under nonreducing conditions was used as a control. The right hemisphere of an end stage prion-affected mouse was fixed in 10% buffered formalin solution. Coronal slices of the brain were cut and immersed in 98% formic acid to reduce infectivity, followed by embedding in paraffin wax. Sections (4 μm) were cut and stained with HE or analyzed by immunohistochemistry. For neuropathological analysis, the lesion profile was determined from the HE-stained sections by scoring the vacuolar changes in nine standard gray-matter areas, as described previously (15). For immunohistochemistry, PrPSc in brain sections was retrieved by the alkaline-based chemical method (16) and then detected using anti-PrP monoclonal antibody SAF84 against the epitope of amino acids 160–170 of the hamster prion protein.