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Volumn 277, Issue 5324, 1997, Pages 381-383

In vitro propagation of the prion-like state of yeast Sup35 protein

Author keywords

[No Author keywords available]

Indexed keywords

PRION PROTEIN;

EID: 0030758280     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.277.5324.381     Document Type: Article
Times cited : (204)

References (27)
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    • 2, 1 mM EDTA, 5% glycerol] containing 1 mM phenylmethylsulfonyl fluoride and protease inhibitor mixture as described [Short Protocols in Molecular Biology, F. M. Ausubel et al., Eds. (Wiley, New York, 1992)]. Cell debris was removed by centrifugation at 15,000g for 20 min.
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    • Paushkin, S.V.1
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    • note
    • Lysates were prepared as described (13), but without addition of protease inhibitors. Each reaction contained 150 μg of total protein and prateinase K (0.4 to 4.0 μg/ml) (Boehringer Mannheim) in a volume of 50 μl. After a 30-min incubation at 37°C, portions (4 μl) were removed and analyzed by protein immunoblotting with antibody to Sup35p as described (6).
  • 19
    • 9844263460 scopus 로고    scopus 로고
    • note
    • psi- (6) or both. Therefore, the extent of conversion reaction was estimated as the Sup35p ratio between cytosolic fraction and sedimented material.
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    • F. E. Cohen et al., Science 264, 530 (1994).
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    • note
    • +] strain was loaded onto a 15 to 40% linear sucrose gradient made in buffer A and centrifuged at 180,000g for 3 hours at 4°C. The gradient was fractionated into 0.4-ml portions. The sedimented material was dissolved in a loading volume of buffer A.
  • 25
    • 9844221481 scopus 로고    scopus 로고
    • note
    • +] with Sup35ΔSp-encoding multicopy plasmid (13) was treated with ribonuclease A (500 g/ml) for 15 min at 2O°C, a high salt concentration (1 M LiCl), and nonionic detergent (1% Triton X-100) for 20 min at 4°C and sedimented by centrifugation through a sucrose layer as described (16). The sedimented material was treated again as above, resuspended in buffer A, made either 1.25 M or 2.5 M with GuHCl, incubated for 30 min at 4°C, precipitated by centrifugation through a sucrose layer, and resuspended in buffer A for use in the conversion reaction.
  • 27
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    • note
    • We thank L. Kisselev, M. Tuite, and I. Stansfield for critical reading of the manuscript and K. Jones for antibody to Sup45p. Supported by grants from INTAS and the Russian Foundation for Basic Research to (M.D.T.-A.) and from the Wellcome Trust to (V.V.K.).


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.