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Volumn 274, Issue 5295, 1996, Pages 2079-2082

Evidence for the conformation of the pathologic isoform of the prion protein enciphering and propagating prion diversity

Author keywords

[No Author keywords available]

Indexed keywords

PRION PROTEIN;

EID: 12644272790     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5295.2079     Document Type: Article
Times cited : (782)

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    • Homogenates (10%, w/v) of human or mouse brain were prepared by repeated extrusion through an 18-gauge syringe needle followed by a 22-gauge needle in phosphate-buffered saline lacking calcium and magnesium ions. For immunoblot analysis, samples were adjusted to 0.5% NP-40 and 0.5% sodium deoxycholate, and samples were digested with proteinase K (PK) (100 μg/ml) for 1 hour at 37°C. Digestion was terminated by the addition of phenylmethylsufonylfluoride (2 mM final concentration) and boiling in electrophoresis sample buffer (3% SDS, 62.5 mM tris, pH 6.8). For deglycosylation, the PK-treated samples were digested for 2 hours with recombinant PNGase F (New England Biolabs) as specified by the supplier, precipitated with four volumes of methanol at -20°C, and resuspended in electrophoresis sample buffer.
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    • note
    • This work was supported by grants from NIH and the American Health Assistance Foundation, as well as by gifts from the Sherman Fairchild Foundation and the Britton Fund. G.T. was supported by a fellowship from an NIH postdoctoral training grant (NS07219).


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