USE OF CAPILLARY SODIUM DODECYL-SULFATE GEL-ELECTROPHORESIS TO DETECTTHE PRION PROTEIN EXTRACTED FROM SCRAPIE-INFECTED SHEEP

Scrapie in sheep and in goats is the prototype of a group of transmiss ible spongiform encephalopathies (TSE). A feature of these diseases is the accumulation in the brain of rod shaped fibrils that form from an aggregated protein that is a protease-resistant form of a modified no rmal host cell protein, In this study, we compared SDS gel capillary e lectrophoresis to conventional SDS-PAGE and Western blot to detect the monomer of this aggregated protein. This prion protein was extracted from the sheep brain by homogenizing the brain stem (10%, w/v) in 0.32 M sucrose and by using a series of ultracentrifugation steps and trea tment with sodium lauroyl sarcosine and proteinase K. After the final centrifugation step, the pellet was resuspended in 0.01 M Tris pH 7.4 in a volume equivalent to 0.1 ml/g of brain used. This resuspended pel let was treated with 1% SDS and 5% 2-mercaptoethanol and boiled for 10 min. The analysis was done in a Beckman P/ACE 5500 using a SDS eel ca pillary (eCap SDS14-200 Beckman capillary). In infected sheep brain sa mples, but not normal sheep, a major peak at a molecular mass of 16.1 kDa and a minor peak with a leading shoulder were observed. Since the molecular mass determined for this protein was lower than that estimat ed on Western blot (22.4 kDa), a Ferguson plot was made to determine i f there were abberations in the molecular mass determination. After co rrection, the major peak was estimated to be 19.2 kDa. This has a bett er correlation with that determined by SDS-PAGE and Western blot. The equivalent amount of brain sample in the capillary was similar to 50 m u g. For Western blot, the amount of brain sample was similar to 20 mg . For this assay, this is similar to 100 times less than that needed f or Western blot for sheep samples.