Polyacrylamide minigels

Western blot analyses was performed on brain tissue collected from one non-inoculated, one TME-infected, and eight BSE-infected cattle: two classified as C-type, four as H-type, and two as L -type. Each sample was separated by SDS-PAGE on 12% polyacrylamide minigels (Invitrogen) and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA) for 45 min at 30 V. The membranes were blocked with 3% bovine serum albumin (BSA) in Tris-buffered saline (TBS) for 1 h and incubated at 4°C overnight with mouse anti-PrP monoclonal antibody 6H4 at a 1:10,000 dilution (0.1 μg/ml) as the primary antibody. Then, a biotinylated sheep anti-mouse secondary antibody at 0.05 μg/ml and a streptavidin–horseradish peroxidase (HRP) conjugate, were used in conjunction with a chemiluminescent detection system (Pierce ECL plus, Thermo Fisher) and visualized on an imaging system capable of detecting luminescence.

Samples of sheep brainstem that had been frozen at -80°C were used to prepare brain homogenate for western blotting. Brain samples were bead homogenized at 20% (w/v) in 1X PBS (Dulbecco’s PBS, pH 7.4, lacking calcium and magnesium) and tested shortly after storage at -80°C. PK digestion of whole brain homogenate was performed in 1X PBS for 1 hour at 37°C, at a final PK of 50 μg/ml, and then each sample was separated by SDS-PAGE on 12% polyacrylamide minigels (Invitrogen) and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA) for 45 min at 30 V. The membranes were blocked with 3% bovine serum albumin (BSA) in Tris-buffered saline (TBS) for 1 h and incubated at 4° C overnight with mouse anti-PrP monoclonal antibody P4 at a 1:10,000 dilution (0.1 μg/ml) as the primary antibody. Then, a biotinylated sheep anti-mouse secondary antibody at 0.05 μg/ml and a streptavidin–horseradish peroxidase (HRP) conjugate were used in conjunction with a chemiluminescent detection system (Pierce ECL plus, Thermo Fisher) and visualized on an imaging system capable of detecting luminescence.

All brain tissues including one sham-inoculated negative control, two TME-infected PRNP knock out cattle, three BSE-infected cattle: two with clinical signs (animal No. 2 and No. 3) and one without clinical signs (animal No. 1) were run on the Western blot (WB). Each sample was digested with 50 µg of proteinase K (PK) for 1 h at 42°C and separated by SDS-PAGE on 12% polyacrylamide minigels (Invitrogen) and then transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) for 45 min at 30 V. Bovine serum albumin (3%) in Tris-buffered saline was used to block the membranes for 1 h and then membrane was incubated with mouse anti-PrP monoclonal antibody 6H4 as the primary antibody at 1:10,000 dilution (0.1 µg/ml) for another hour. Then, a biotinylated sheep antimouse secondary antibody at 0.05 µg/ml and a streptavidin–horseradish peroxidase conjugate were used in conjunction with a chemiluminescent detection system (Pierce ECL plus, Thermo Fisher) and visualized on an imaging system capable of detecting luminescence.