Name: protein misfolding cyclic amplification of prions
Uploaded: 2012-11-07T12:00:00
Description: Watch this Scientific Journal Video about Protein Misfolding Cyclic Amplification of Prions at JoVE.com

Nous tenons à remercier le Dr Vesper Fe Marie Ramos pour la lecture critique du manuscrit. Ce travail a été soutenu par le National Center for Research Resources (P20 RR0115635-6, C06-01 et RR17417 G20RR024001) et l&#39;Institut national des troubles neurologiques et des maladies (2R01 NS052609).

1. Préparation de l&#39;équipement <ol><li> Utilisez un 3000 Misonix ou Misonix 4000 sonicateur (Farmingdale, NY) relié à un bain Thermo Electron Neslab EX-7 de l&#39;eau (Newington, NH) pour maintenir une température constante de 37 ° C. Soniquer les échantillons dans 200 pi à parois minces barrettes de tubes PCR avec bouchons bombés obtenus à partir de Thermo Scientific (Waltham, MA). </li><li> Un appareil à ultrasons nouvelle marque nécessite un &quot;break-in&quot; période de fonctionnement continu <s...

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzymatic+treatment+of+soil-bound+prions+effectively+inhibits+replication.">An enzymatic treatment of soil-bound prions effectively inhibits replication.</a> Appl. Environ. Microbiol. 77, 4313-4317 (2011).</li><li>Saunders, S. E., Shikiya, R. A., Langenfeld, K., Bartelt-Hunt, S. L., Bartz, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+efficiency+of+soil-bound+prions+varies+with+soil+type.">Replication efficiency of soil-bound prions varies with soil type.</a> Journal of virology. , (2011).</li><li>Schutt, C. R., Bartz, J. 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Les défis de amplification protéines prions infectieuses sont les longues périodes d&#39;incubation et les frais de dans des expériences in vivo. La technique PMCA est un moyen rentable pour amplifier les prions infectieux. Plusieurs laboratoires ont confirmé la capacité de PMCA avec précision amplifier souches de prion in vitro 7, 9, 12, 14, 19,24. Les maladies à prions peuvent être transmis entre les espèces. Bessen et Marsh ont effectivement...

<table border="1"><tbody><tr><td> Réactif / équipement </td><td> Fabricant </td><td> Cat. Nombre </td></tr><tr><td> Misonix 3000 </td><td> Misonix </td><td> S-3000 </td></tr><tr><td> Misonix 4000 </td><td> Misonix </td><td> S-4000 </td></tr><tr><td> Grinder tissus Tenbroeck </td><td> Kontes </td><td> 885000-0007 </td></tr><tr><td> Bain Neslab EX-7 Eau </td><td> Thermo Electron </td><td> Neslab EX-7 </td></tr><tr><td> 0,2 ml Bandes de tubes PCR </td><td> Thermo Scientific </td><td> AB-0451 </td></tr><tr><td> Triton X-100 </td><td> Sigma Aldrich </td><td> T9284-100ML </td></tr><tr><td> Inhibiteur de la protéase complète </td><td> Roche </td><td> 11 697 498 001 </td></tr><tr><td> EDTA </td><td> JT Baker </td><td> 4040-00 </td></tr><tr><td> DPBS </td><td> Mallinckrodt Baker Mediatech </td><td> 21-031-CV </td></tr><tr><td> Soakers Lab Versi-Dry </td><td> Fisher Scientific </td><td> 14 206 28 </td></tr><tr><td> Repti Therm Chauffage </td><td> Zoo Med Laboratories, Inc </td><td> RH-4 </td></tr></tbody></table>

protein misfolding cyclic amplification of prions

Les prions sont des agents infectieux qui causent l&#39;encéphalopathie spongiforme inévitablement fatale transmissible (EST) chez les animaux et les humains 9,18. La protéine prion présente deux isoformes distincts, le non-infectieuse codée par l&#39;hôte de protéine (PrP C) et la protéine infectieuse (PrP Sc), une isoforme anormale pliée de PrP C 8. L&#39;un des défis de travailler avec les prions est la longue période d&#39;incubation avant l&#39;apparition des signes cliniques qui suivent le 13 inoculation d&#39;accueil. Cette traditionnellement chargé longs et coûteux essais biologiques d&#39;origine animale. En outre, les propriétés biochimiques et biophysiques de la PrP Sc sont mal caractérisées en raison de leur conformation inhabituelle et états d&#39;agrégation. PrP Sc peut engendrer la conversion de PrP C en PrP Sc in vitro 14. PMCA est une technique in vitro qui prend avantage de cette capacité en utilisant les cycles de sonication et d&#39;incubation pour produire de grandes quantités de PrP Sc, à une vitesse accélérée, à partir d&#39;un système contenant des quantités excessives de PrP C et des quantités infimes de la graine PrP Sc 19. Cette technique s&#39;est avérée efficace pour résumer la spécificité d&#39;espèce et la souche de PrP Sc conversion de PrP C, à imiter les interférences souche de prion, et d&#39;amplifier de très faibles niveaux de PrP Sc dans les tissus infectés, fluides, et les échantillons environnementaux 6,7,16, 23. Cet article détaille le protocole de PMCA, y compris des recommandations pour minimiser la contamination, des résultats constants, et de quantifier ces résultats. Nous discutons également plusieurs applications PMCA, notamment la production et la caractérisation des souches de prions infectieux, les interférences souche de prion, et la détection des prions dans l&#39;environnement.

Amplification cyclique protéine repliement (PMCA) est utilisée pour amplifier la PrP Sc in vitro 7, 12, 14, 19, 24. Un succès PrP Sc amplification est démontré par une augmentation de l&#39;intensité des bandes sur Western blot de la protéine prion PK-résistante (migration entre 19 et 30 kDa pour souches de prion de hamster dérivés), comme illustré à la figure 3. L&#39;augmentation de l&#39;intensité de la bande après PMCA indique l&#39;amplificati...

Watch this Scientific Journal Video about Protein Misfolding Cyclic Amplification of Prions at JoVE.com

Protein Misfolding Cyclic Amplification of Prions

Repliement des protéines amplification cyclique (PMCA) est un test in vitro pour l&#39;étude de la conversion du prion et les obstacles souche et l&#39;espèce. Il peut également être utilisé comme un test de détection de prion.

Protein Misfolding Cyclic Amplification des prions

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

Research

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