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

We willen graag Dr Vesper Fe Marie Ramos bedanken voor het kritisch lezen van het manuscript. Dit werk werd ondersteund door de National Center for Research Resources (P20 RR0115635-6, C06 RR17417-01 en G20RR024001) en het Nationaal Instituut voor Neurologische Aandoeningen en Stroke (2R01 NS052609).

1. Voorbereiden van de Apparatuur <ol><li> Gebruik Misonix 3000 of 4000 Misonix sonicator (Farmingdale, NY) verbonden met een Thermo Electron Neslab EX-7 waterbad (Newington, NH) om een ​​constante temperatuur te houden van 37 ° C. Sonificeer de monsters in 200 ul dunwandige buis PCR strips met gewelfd deksel verkregen Thermo Scientific (Waltham, MA). </li><li> Een gloednieuwe sonicator vereist een &quot;break-in&quot;-periode van continue werking 9. Een twee maanden break-in periode bestaat uit een 40...

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Uitdagingen van het amplificeren besmettelijke prion-eiwitten zijn de lange incubatietijd en de kosten van de in vivo experimenten. De PMCA techniek is een kosteneffectieve manier om besmettelijke prionen agenten te versterken. Verschillende laboratoria hebben het vermogen van PMCA nauwkeurig prion stammen amplificeren in vitro 7, 9, 12, 14, 19,24. Prionziekten worden overgedragen tussen soorten. Bessen en Marsh effectief hebben geënt hamsters met overdraagbare ...

<table border="1"><tbody><tr><td> Reagens / Equipment </td><td> Fabrikant </td><td> Cat. Aantal </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> Tenbroeck Tissue Grinder </td><td> Kontes </td><td> 885000-0007 </td></tr><tr><td> Neslab EX-7 Water Bath </td><td> Thermo Electron </td><td> Neslab EX-7 </td></tr><tr><td> 0,2 ml PCR Tube Strips </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> Compleet Proteaseremmer </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 tot 031-CV </td></tr><tr><td> Versi-Dry Lab soakers </td><td> Fisher Scientific </td><td> 14 206 28 </td></tr><tr><td> Repti Therm Heater </td><td> Zoo Med Laboratories, Inc </td><td> RH-4 </td></tr></tbody></table>

protein misfolding cyclic amplification of prions

Prionen zijn infectieuze agentia die de onvermijdelijk fatale overdraagbare spongiforme encefalopathie (TSE) veroorzaken bij dieren en mensen 9,18. De prion eiwit heeft twee verschillende isovormen, niet-infectieuze gastheer gecodeerd eiwit (PrP C) en infectieus eiwit (PrP Sc), een abnormaal gevouwen isovorm van PrP C 8. Een van de uitdagingen van het werken met prion middelen is de lange incubatietijd voorafgaand aan de ontwikkeling van klinische tekenen die volgden gastheer inoculatie 13. Dit traditioneel opdracht lange en dure dieren bioassay studies. Verder zijn de biochemische en biofysische eigenschappen van PrP Sc slecht vanwege hun bijzondere conformatie en aggregatietoestanden gekarakteriseerd. PrP Sc kan zaad de omzetting van PrP C PrP Sc in vitro 14. PMCA een in vitro techniek die wordt Advantage van dit vermogen gebruik sonificatie en incubatie cycli om grote hoeveelheden PrP Sc produceren hogere snelheid, een systeem met overmaat PrP C en kleine hoeveelheden van het PrP Sc seed 19. Deze techniek heeft bewezen effectief te recapituleren de soort en stam specificiteit van PrP Sc omzetting van PrP C tot prionstam interferentie emuleren, en zeer lage niveaus van PrP Sc amplificeren van geïnfecteerde weefsels, vloeistoffen en milieumonsters 6,7,16, 23. Dit document beschrijft de PMCA protocol, met inbegrip van aanbevelingen voor het minimaliseren van verontreiniging, het genereren van consistente resultaten, en kwantificeren van die resultaten. We bespreken ook verschillende PMCA toepassingen, inclusief productie en karakterisering van besmettelijke prion stammen prionstam interferentie en de detectie van prionen in het milieu.

Protein misfolding cyclische amplificatie (PMCA) wordt gebruikt om PrP Sc in vitro 7, 12, 14, 19, 24 amplificeren. Een succesvolle PrP Sc amplificatie wordt aangetoond door een verhoging in bandintensiteit op Western blots van de PK-resistente prioneiwit (migreren tussen 19 en 30 kDa voor hamster prion afgeleide stammen) zoals getoond in figuur 3. De toename van de band intensiteit na PMCA geeft amplificatie van het PK-resistente PrP Sc materiaal. Suc...

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

Protein Misfolding Cyclic Amplification of Prions

Protein misfolding cyclische amplificatie (PMCA) is een in vitro assay voor de studie van prion conversie en spanning en species barrières. Het kan ook worden gebruikt als een prion detectie assay.

Eiwit misfolding Cyclische Versterking van prionen

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

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