Western Blots

Samples were digested with PK and then analyzed by western blotting using the monoclonal antibodies SHa31 or P4 as described previously.²³ Briefly, samples were digested with 5, 10 or 50 μg/ml PK in the presence of 0.045% (w/v) SDS for 1 h at 37°C. Samples were boiled in 1x NuPAGE SDS-PAGE sample buffer for 5 min, and then an equivalent to 5 μl of the sPMCA reaction products was electrophoresed on 12% (w/v) polyacrylamide gels (precast NuPAGE SDS-PAGE Bis-Tris; Invitrogen). Samples were transferred to polyvinylidene difluoride membranes by electroblotting and then blocked in 3% (w/v) skimmed milk-PBS. Blots were probed with SHa31 or P4 monoclonal antibody at a dilution of 1/80,000 for SHa31(Cayman Chemicals) or 1/1,000 for P4 (R-Biopharm) in 0.5% (w/v) skimmed milk-PBS. After washing, the blots were probed using a secondary goat anti-mouse horseradish peroxidase (HRP) conjugate at a 1/20,000 dilution (Dako). Blots were visualized using an HRP chemiluminescent substrate (Geneflow) and a Photek imaging system.

For densitometry, gel images were measured with ImageJ software and the lane pixel densities were plotted, the signal maxima for each band was used (molecular weight analysis) or the total areas corresponding to the band peaks were defined and the background for each lane was subtracted (all other analysis). When calculating molecular weights, the migration distance of proteins through the SDS-PAGE gel was measured and a standard curve generated using the migration distances of the 60, 50, 40, 30 and 20 kDa molecular mass standards (NuPAGE Magicmark, Invitrogen). Using linear regression analysis, the molecular weight of unglycosylated PrP^Sc was then calculated from the curves using Graphpad prism software. The SHa31/P4 ratio for a sample was calculated as the ratio of the western blot signals produced by samples when probed with SHa31 and P4 monoclonal antibodies on 2 separate blots. This is a measure of the presence of the P4 epitope within PrP^Sc after PK digestion, the epitope is present at relatively high levels in ovine scrapie isolates and H-BSE, but at much lower levels in C-BSE and L-BSE isolates.²⁶ The ratio of PK-resistant PrP^Sc when samples were digested at 10 and 50 μg/ml PK were determined using densitometry to determine total PrP^Sc signals as described above.