Purification of the scrapie agent by density gradient centrifugation

J Gen Virol. 1984 Feb:65 ( Pt 2):415-21. doi: 10.1099/0022-1317-65-2-415.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Brain / microbiology*
  • Brain / ultrastructure
  • Cell Membrane / microbiology
  • Cell Membrane / ultrastructure
  • Centrifugation, Density Gradient / methods
  • Cricetinae
  • Male
  • Neurons / microbiology*
  • Prions / isolation & purification*
  • Prions / ultrastructure
  • Retina / microbiology*

Substances

  • Prions