Purification of the scrapie agent by density gradient centrifugation.
Plasma membrane-enriched preparations from scrapie-infected and healthy hamster brains, as well as preparations of neural retina, were sonicated, then separated by rate-zonal sedimentation in 10 to 25% Nycodenz gradients. Gradient fractions were extracted with 0.5% Triton X-100 and re-fractionated by equilibrium density centrifugation in linear 25 to 40% CsCl gradients. Infectivity was highest in a fraction having a density of 1.280 g/ml and which contained a visible band of material. Digestion of the Nycodenz fractions with proteinase K before detergent extraction and CsCl fractionation resulted in a shift in the visible band to a density of 1.235 g/ml with most of the scrapie infectivity remaining at 1.280 g/ml. When labelled with 125I after 40-fold concentration, this 1.280 g/ml CsCl fraction from the proteinase K-treated gradients contained only a single band of protein(s) having a mol. wt. near 30 000. No differences were seen between proteins in healthy or scrapie-infected preparations.
D000818:Animals; D001921:Brain / Q000382:microbiology* / Q000648:ultrastructure; D002462:Cell Membrane / Q000382:microbiology / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient / Q000379:methods; D006224:Cricetinae; D008297:Male; D009474:Neurons / Q000382:microbiology*; D011328:Prions / Q000302:isolation & purification* / Q000648:ultrastructure; D012160:Retina / Q000382:microbiology*
D011328:Prions