Comparison of PrP
and total protein during the enrichment process. A) Western blots depicting the migration pattern of PrPSc in the final enriched pellet (P) and in the supernatants after the precipitation (W1) and the wash (W2) steps in native sheep scrapie. Total proteins in W1 and W2 were precipitated with 9 volumes of methanol over night at -70°C. SAL1 (1:3000) was used as the primary antibody and alkaline phosphatase labeled goat anti-rabbit IgG (1:1500, Roche) as the secondary antibody. Each lane contains 3 mg brain equivalent. The table shows percentage recoveries of PrPSc in P, W1 and W2. The estimations were made by first determining the values for integrated optical density (IOD) due to PrPSc for each lane. The sum of the values in P, W1 and W2 was then taken to be one hundred percent total PrPSc in the starting material. Values due to PrPSc in each of P, W1 and W2 were then deduced as percentages of the sum total as described in Materials and Methods. Proteinase K used was 30 μg per ml homogenate. B) Silver staining of H (starting undigested homogenate), P, W1 and W2 run on a SDS-PAGE system. All lanes contain 3 mg brain equivalent loads except for lane H which contains 0.3 mg of the same. Molecular weight markers are shown in lane M. It may be noticed that the some of the proteins present in H are also present in W1, in the discarded supernatant after the precipitation step. Lanes P and W2 contain few detectable proteins. The table provides a quantitative estimation of the degree of purification of PrPSc in P after taking into account, signals due to total proteins in lanes H and P respectively.