Name: isolation of soluble and insoluble prp oligomers in the normal human brain
Uploaded: 2012-10-03T12:00:00
Description: Watch this Scientific Journal Video about Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain at JoVE.com

Den Forfatterne er taknemmelige for den menneskelige hjerne og Spinal Fluid Resource Center (Los Angeles, CA) til at tilvejebringe normale hjerne prøver. Denne undersøgelse blev støttet af National Institutes of Health (NIH) R01NS062787, CJD Foundation, Alliance biologisk sikre, samt University Center on Aging og sundhed med støtte fra McGregor Foundation og præsidentens Skønsmæssig Fund (Case Western Reserve University) .

1. Fremstilling af hjernehomogenat og detergent-opløselige (S2) og-Uopløseligt (P2) Fraktioner <ol><li> Tag 100 mg frosset humant hjernevæv, og der tilsættes 100 pi lysisbuffer (10 mM Tris, 150 mM NaCl, 0,5% Nonidet P-40 (NP-40), 0,5% EDTA, pH 7,4). </li><li> Homogenisere den på is med pistil drevet af en batteridrevet motor, fryse den i tøris i 5 minutter og homogeniseres igen med pistil drevet af hænder først og derefter ved motoren, og der tilsættes 300 gl eller 800 pi lysisbuffer (dette er enten 20 % elle...

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Kombinationen af fremgangsmåder rapporteret her isolerer konsekvent uopløselige PrP ° aggregater og opløselige PrP ° oligomerer fra den normale menneskelige hjerne. Ultracentrifugering ved 100.000 x g i en time er en klassisk metode, der har været meget anvendt til separation af det uopløselige PrP Sc fra den opløselige PrP C 14. Selv om det er effektivt, en af ​​forholdsreglerne er at undgå forurening under trække supernatanten (S2) efter centrifugering. Da gele...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer (valgfrit) </td></tr><tr><td> Phenylmethylsulfonylfluorid </td><td> Sigma-Aldrich </td><td> P7626 </td><td> 72 mM i 2-propanol </td></tr><tr><td> Proteinase K </td><td> Sigma-Aldrich </td><td> P2308 </td><td> 2 mg / ml i H2O </td></tr><tr><td> PNGase F </td><td> New England Biolabs </td><td> MWGF200 </td><td></td></tr><tr><td> Ultracentrifuge LE-80K, SW55 rotor </td><td> Beckman Coulter </td><td> Part No 365.668, 356.860 </td><td></td></tr><tr><td> Superdex 200 HR perler </td><td> GE Healthcare </td><td> 17-1088-01 </td><td></td></tr><tr><td> AKTA FPLC-system </td><td> GE Healthcare </td><td> 18-1900-26 </td><td></td></tr><tr><td> Molekylvægtmarkører </td><td> Sigma-Aldrich </td><td> MWGF200 </td><td> Varierede </td></tr><tr><td> Dynabeads M-280 magnetiske perler </td><td> Invitrogen </td><td> 143-01 </td><td></td></tr><tr><td> 3F4 antistof </td><td> Covance </td><td> SIG-39.600 </td><td></td></tr><tr><td> 1E4-antistof </td><td> Cell Sciences </td><td> M1840 </td><td></td></tr><tr><td> Klar gel 15% Tris-HCI forstøbte geler </td><td> Bio-Rad </td><td> 345-0020 </td><td></td></tr></tbody></table>

isolation of soluble and insoluble prp oligomers in the normal human brain

Den centrale hændelse i patogenesen af prionsygdomme involverer en omdannelse af værten-kodede cellulære prionprotein PrP ° i dets patogene isoform PrP Sc 1. PrP C er detergent-opløselig og følsom over for proteinase K (PK)-fordøjelse, mens PrPsc danner detergent-uopløselige aggregater, og er delvis resistente over for PK 2-6. Omdannelsen af PrP ° til PrPsc er kendt for at involvere en konformationel forandring af α-helical til β-sheet struktur af proteinet. Imidlertid er in vivo-reaktionsvej stadig dårligt forstået. En foreløbig endogen PrP Sc, mellemliggende PrP * eller &quot;silent prion&quot;, har endnu ikke identificeret i den uinficerede hjerne 7. Ved hjælp af en kombination af biofysiske og biokemiske metoder, vi identificerede uopløselige PrP C aggregater (betegnet iPrP C) fra inficerede pattedyr hjerner og dyrkede neuronalceller 8, 9. Her beskriver vi detaljerede procedurer ved disse metoder, herunder ultracentrifugering i detergent buffer, sucrose trinvis gradient sedimentation, størrelseseksklusion kromatografi, iPrP berigelse af gen 5-protein (G5p) som specifikt binder til strukturelt ændrede PrP former 10 og PK-behandling. Kombinationen af disse fremgangsmåder isolerer ikke bare uopløselige PrPsc og PrP ° aggregater, men også opløselige PrP ° oligomerer fra den normale menneskelige hjerne. Eftersom protokollerne beskrevet her har været anvendt til at isolere både PrP Sc fra inficerede hjerner og iPrP C fra ikke-inficerede hjerner, de giver os mulighed for at sammenligne forskelle i fysisk-kemiske egenskaber, neurotoksicitet og infektivitet mellem de to isoformer. En sådan undersøgelse vil i høj grad forbedre vores forståelse af de infektiøse proteinholdige patogener. Fysiologi og patofysiologi iPrP C er uklart på nuværende tidspunkt. Især i en nyligt identified mennesker prionsygdom betegnes trinløst proteasefølsomme prionopathy, fandt vi en ny PrPsc, der deler den immunoreaktive adfærd og fragmentering med iPrP C 11, 12. Desuden har vi for nyligt vist, at iPrP C er det vigtigste arter, som vekselvirker med amyloid-β-protein i Alzheimers sygdom 13. I den samme undersøgelse blev disse fremgangsmåder anvendt til at isolere Abeta aggregater og oligomerer ved Alzheimers sygdom 13, tyder deres anvendelse til ikke-prion-proteinaggregater involveret i andre neurodegenerative lidelser.

Watch this Scientific Journal Video about Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain at JoVE.com

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

En ny art af cellulær prionprotein (PrP<sup&gt; C</sup&gt;) Er for nylig blevet identificeret i uinficerede humane hjerner under anvendelse af fremgangsmåderne beskrevet her. Disse fremgangsmåder kan anvendes til at isolere forskellige PrP arter, mens nogle af dem er også anvendelige til isolering af andre fejlfoldede proteinaggregater fra humane hjerner.

Isolering af opløselige og uopløselige PrP Oligomerer i normal human hjerne

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform ...

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