Name: chromatographic purification of highly active yeast ribosomes
Uploaded: 2011-10-24T12:00:00
Description: Watch this Scientific Journal Video about Chromatographic Purification of Highly Active Yeast Ribosomes at JoVE.com

Vi ønsker at takke alle medlemmerne af Dinman laboratoriet, herunder Ashton Belew, Karen Jack, Sharmishtha Musalga, Sergey Sulima, Shivani Reddy, og Michael Rhodin for deres hjælp og input på dette projekt. Dette arbejde blev støttet af tilskud til AM fra American Heart Association (AHA 0630163N) og JDD fra National Institutes of Health (5R01 GM058859-12). JAL blev understøttet af en amerikansk Reinvestering og Recovery Act of 2009 supplement til den forælder, tilskud (3R01GM058859-11S1).

1. Udarbejdelse af en cystein opladet Sulfolink harpiks <ol><li> Forbered Sulfolink resin (Pierce) ved stuetemperatur. </li><li> Placer en i alt 10 ml af en 50% opslæmning af Sulfolink kobling gel som leveret af fabrikanten i to 10 ml plastik hætteglas med 5 ml fordeles i hvert hætteglas. </li><li> Kort centrifuge hætteglas ved 850 xg og fjern forsigtigt supernatanter. </li><li> Vask gel tre gange i kobling buffer (50 mM Tris, pH 8,5, 5 mM EDTA) ved opblandes perler i 5 ml, centrifugering kortvarigt ved 850 xg og ...

<ol>
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Ribosom rensning protokoller hovedsagelig tale lysering celler, høst et cytosol fraktion fra en lav hastighed spin, og derefter pelletering ribosomer ved høj hastighed centrifugering 1. Mens et par nye metoder har været brugt til rensning af bakterielle ribosomer, havde det samme ikke været tilfældet for eukaryoter 2-4. Selv om yderligere skridt er blevet tilføjet langs år, fx salt vasker, og glycerol hynder (se f.eks 5), har biokemiske og strukturelle studier af gær ribosomer bl...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td></tr><tr><td> Sulfolink Kobling harpiks </td><td> Pierce </td><td> 20402 </td></tr><tr><td> Mini perle-beater 16 </td><td> Biospec </td><td> 607 </td></tr><tr><td> Optima Max E ultracentrifuge </td><td> Beckman Coulter </td><td></td></tr><tr><td> Legend RT </td><td> Sorvall </td><td></td></tr><tr><td> 0,5 mm Glasperler </td><td> Biospec </td><td> 11079105 </td></tr><tr><td> Tris </td><td> Sigma-Aldrich </td><td> 252859 </td></tr><tr><td> EDTA </td><td> Sigma-Aldrich </td><td> E9884 </td></tr><tr><td> DTT </td><td> Sigma-Aldrich </td><td> 43815 </td></tr><tr><td> HEPES </td><td> Sigma-Aldrich </td><td> 54459 </td></tr><tr><td> NH 4 Cl </td><td> Sigma-Aldrich </td><td> A9434 </td></tr><tr><td> KCl </td><td> Sigma-Aldrich </td><td> 60128 </td></tr><tr><td> Mg (OAC) 2 </td><td> Sigma-Aldrich </td><td> M5661 </td></tr><tr><td> KOH </td><td> Sigma-Aldrich </td><td> 221473 </td></tr><tr><td> Glycerol </td><td> Sigma-Aldrich </td><td> G5516 </td></tr><tr><td> Puromycin </td><td> Sigma-Aldrich </td><td> P7255 </td></tr><tr><td> GTP </td><td> Fermentas </td><td> R0461 </td></tr></tbody></table>

chromatographic purification of highly active yeast ribosomes

Eukaryote ribosomer er meget mere labile i forhold til deres eubacterial og archael modstykker, og udgør derfor en stor udfordring for forskerne. Særligt generende er, at lyse af celler frigiver en lang række proteaser og nucleaser som kan forringe ribosomer. Derfor er det vigtigt at adskille ribosomer fra disse enzymer så hurtigt som muligt. Desværre, konventionelle forskellen ultracentrifugering metoder blade ribosomer udsat for disse enzymer til uacceptabelt lange perioder, påvirker deres strukturelle integritet og funktionalitet. For at løse dette problem, vi benytter en kromatografisk metode ved hjælp af en cystein opkrævet Sulfolink harpiks. Denne enkle og hurtige anvendelse reducerer co-rensende proteolytiske og nucleolytic aktiviteter, der producerer høje udbytter af intakt, meget biokemisk aktiv gær ribosomer. Vi foreslår, at denne metode også bør gælde for pattedyr ribosomer. Den enkelhedmetoden, og de forbedrede renhed og aktivitet chromatografisk renset ribosom repræsenterer et betydeligt teknisk fremskridt for studiet af eukaryote ribosomer.

Watch this Scientific Journal Video about Chromatographic Purification of Highly Active Yeast Ribosomes at JoVE.com

Chromatographic Purification of Highly Active Yeast Ribosomes

Forurening af præparater af eukaryote ribosomer renses ved traditionelle metoder ved co-rensende nucleaser og proteaser negativt virkninger på downstream-biokemiske og strukturelle analyser. En hurtig og enkel kromatografisk oprensning metoden anvendes til at løse dette problem ved hjælp af gær ribosomer som model system.

Chromatografisk Rensning af højaktiv Gær ribosomer

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

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