FIG. 2.
Detection of protease-resistant PrPSc within CNS and mammary gland tissues of animals displaying clinical scrapie. Tissues were prepared as 10% or 40% (wt/vol) homogenates for spinal cord and mammary gland tissue, respectively, as described previously elsewhere (15). Native or proteinase K-digested homogenates (25 μg/ml; 1 h at 37°C) were analyzed as indicated. Protease-resistant PrPSc was readily detectable within spinal cord tissue (SC; lanes 1 to 2) but was not detectable within mammary gland samples (MG; lanes 3 to 6). Either 0.33 mg (0350/07) or 0.165 mg (0326/07 and 0344/07) of spinal cord tissue and 1.32 mg (lanes 3 and 4) and 6 mg (lane 5) of mammary gland tissue was analyzed per lane. Protease-resistant PrPSc was still undetectable from 20 mg of mammary gland tissue following precipitation with sodium phosphotungstic acid (25) prior to analysis (lane 6). PrPSc within scrapie-positive brain tissue (63 μg) was readily detected by this method after spiking into 20 mg of mammary gland homogenate (B, lane 7). Full-length and fragmented protease-sensitive PrPC was readily detected within mammary gland tissue (lane 3). Animal numbers are indicated. PrP was detected with monoclonal antibody SHA31; molecular mass markers are indicated (kDa).