Name: a step-by-step method for the reconstitution of an abc transporter into nanodisc lipid particles
Uploaded: 2012-08-31T12:00:00
Description: Watch this Scientific Journal Video about A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles at JoVE.com

Questo lavoro è stato sostenuto dal Canadian Institute of Health Research. CSC è stato finanziato da una borsa di studio post-dottorato della scienze naturali e ingegneria Research Council del Canada. FD è un Tier II Canada Research Chair.

<html> Ricostituzione del processo globale Il processo inizia ricostituzione miscelando la membrana proteina scaffold (MSP) con il complesso purificato MalFGK 2 in presenza di detergente-solubilizzate fosfolipidi. La fase è seguita dalla rimozione lenta del detergente da un materiale adsorbente polistirene chiamato Bio-Beads o Amberlite (Figura 1). L&#39;auto-assemblaggio processo si verifica molto probabilmente a causa delle interazioni tra i fosfolipidi apolari idr...

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Descriviamo una semplice procedura per la ricostituzione del trasportatore maltosio in nanodiscs. Il trasportatore è ATPasi attiva e l&#39;interazione con il maschio solubile partner di legame può essere ricreato (Figura 3). La ricostituzione di successo del trasportatore in nanodiscs aprire la strada a ulteriori analisi biofisica e biochimica. Di particolare interesse sarà l&#39;analisi sistematica della ATPasi MalK e attività di trasporto in maltosio detersivo, liposomi e nanodiscs. Trasportatori ...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Amicon Ultra-4 50K filtro centrifugo </td><td> Millipore </td><td> UFC805008 </td><td> Segui protocollo del produttore per un uso corretto </td></tr><tr><td> Bio-Beads SM-2 Adsorbente </td><td> Bio-Rad </td><td> 152-3920 </td><td></td></tr><tr><td> E. coli lipidi totali </td><td> Avanti Polar Lipids </td><td> 100500C </td><td> Disciolto in cloroformio, come appropriato per gestire un solvente organico </td></tr><tr><td> Ni sefarosio HP resina </td><td> GE Healthcare </td><td> 17-5268-01 </td><td></td></tr><tr><td> Fosforo soluzione standard </td><td>Sigma-Aldrich </td><td> P3869 </td><td></td></tr><tr><td> pMSP1D1 </td><td> Addgene </td><td> 20061 </td><td></td></tr><tr><td> Superdex 200 HR 10/300 </td><td> GE Healthcare </td><td> 17-5172-01 </td><td></td></tr><tr><td></td><td></td><td></td><td> Tabella I. reagenti specifici. </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> Nome </td><td> Composizione </td><td> Commenti </td></tr><tr><td> DDM magazzino </td><td> 10% w / v DDM </td><td> Risospendere in acqua MilliQ e conservare a -20 ° C </td></tr><tr><td> MalFGK 2 stock </td><td> 1-2 mg / ml 50 mM Tris-HCl, pH7.9 100 mM NaCl 10% v / v glicerolo 0,03% w / v DDM </td><td> Conservare a -70 ° C dopo purificazione </td></tr><tr><td> MSP magazzino </td><td> 10-15 mg / ml 50 mM Tris-HCl, pH7.9 100 mM NaCl 10% v / v glicerolo </td><td> Conservare a -70 ° C dopo la purificazione nel &lt;aliquote da 1 ml e di evitare eccessivi cicli gelo / disgelo </td></tr><tr><td> Fosfolipidi magazzino </td><td> 5 nM E. coli totale lipidi 0,5% w / v (10 mM) DDM 50 mM Tris-HCl, pH 7,9 50 mM NaCl </td><td> Conservare a 4 ° C per 1 settimana </td></tr><tr><td> TS tampone </td><td> 50 mM Tris-HCl, pH 7,9 50 mM NaCl </td><td> Conservare a 4 ° C </td></tr><tr><td> TSG10 tampone </td><td> 50 mM Tris-HCl, pH7.9 100 mM NaCl 10% v / v glicerolo </td><td> Conservare a 4 ° C </td></tr><tr><td> TSG20 tampone </td><td> 50 mM Tris-HCl, pH 8 100 mM NaCl 20% v / v glicerolo </td><td> Conservare a 4 ° C </td></tr><tr><td> TSGD tampone </td><td> 50 mM Tris-HCl, pH7.9 100 mM NaCl 10% v / v glicerolo 0,03% w / v DDM </td><td> Conservare a 4 ° C e aggiungere DDM poco prima dell&#39;uso </td></tr><tr><td colspan="3"> Tabella II. Soluzione ricette. </td></tr></tbody></table></td></tr></tbody></table>

a step-by-step method for the reconstitution of an abc transporter into nanodisc lipid particles

Il nanodisc è una particella discoidale (~ 10-12 nm di grandi dimensioni) che le proteine ​​di membrana trappola in una piccola zona di doppio strato di fosfolipidi. Il nanodisc è un&#39;opzione particolarmente interessante per studiare proteine ​​di membrana, in particolare nel contesto delle interazioni ligando-recettore. Il metodo introdotto da Sligar e colleghi si basa sulle proprietà di un anfipatiche altamente ingegnerizzato ad a-elica proteina scaffold derivato dal apolipoproteina A1. Le facce idrofobe della proteina scaffold interagire con i grassi acil catene laterali del doppio strato lipidico che le regioni polari affrontare l&#39;ambiente acquoso. Analisi di proteine ​​di membrana in nanodiscs hanno vantaggi significativi rispetto liposoma perché le particelle sono piccole, omogenea e idrosolubile. Inoltre, metodi biochimici e biofisici normalmente riservati alle proteine ​​solubili possono essere applicati, e da entrambi i lati della membrana. In questo protocollo visiva, presentiamo un passo-passo ricostituzione di un carattere benterizzato trasportatore batterica ABC, il maschio-MalFGK 2 complessi. La formazione del disco è un processo di auto-assemblaggio che dipende da interazioni idrofobiche che avvengono durante la rimozione progressiva del detersivo. Si descrivono le fasi essenziali e sottolineano l&#39;importanza di una corretta scelta proteina-lipide rapporto al fine di limitare la formazione di aggregati e grandi polidispersi liposoma-come particelle. Qualità semplici controlli, come la cromatografia di gel filtrazione, elettroforesi su gel nativo e dinamico spettroscopia di dispersione della luce in modo che i dischi sono stati adeguatamente ricostituito.

Watch this Scientific Journal Video about A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles at JoVE.com

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

Nanodiscs sono piccole particelle discoidali che incorporano proteine ​​di membrana in una piccola zona di doppio strato di fosfolipidi. Forniamo un protocollo visivo che mostra il passo-passo incorporazione del trasportatore MalFGK2 in un disco.

A Step-by-step Metodo per la ricostituzione di un Transporter ABC in particelle lipidiche Nanodisc

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a ...

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