Name: in vitro analysis of pdz-dependent cftr macromolecular signaling complexes
Uploaded: 2012-08-13T13:00:00
Description: Watch this Scientific Journal Video about In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes at JoVE.com

Vårt arbete har finansierats med bidrag från American Heart Association (Greater sydöstra Affiliate) Början-bidrag-i-bistånd 0765185B, Elsa U. Pardee Foundation forskningsanslag, och Wayne State University intramural start fonden och Hjärt Forskningsinstitut Isis Initiative utmärkelse. Denna metod för in vitro-CFTR makromolekylära komplex sammansättning ursprungligen uppfunnen av Dr AP Naren (University of Tennessee Health Science Center).

1. Expression och rening av rekombinanta taggade fusionsproteiner i bakterier <ol><li> Amplifiera definierade områden av den C-svansar (de sista 50-100 aminosyror innehållande de PDZ-motiv vid C-terminalen) för CFTR, 2 LPA, MRP2, MRP4, β 2 pa, och NHERFs (full längd eller PDZ1 eller PDZ2 domäner ) av PCR-metoden. </li><li> Klona PCR-produkter in pGEX4T-1-vektorn för GST-fusionsproteiner (såsom GST-NHERFs, GST-MRP4 CT), pMAL-c2-vektorn för MBP-fusionsproteiner (såsom MBP-β 2 ...

<ol>
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I detta protokoll vi visat ett förfarande för in vitro montering och detektering av en CFTR-innehållande makromolekylärt komplex signalering genom att använda renade proteiner (eller proteinfragment) och / eller cellysat som rapporterats tidigare 16-19,29,30. För att uppnå bästa resultat följande kritiska punkter under beredningsprocessen kräver särskild uppmärksamhet: <ul><li> Det är viktigt att pH för elueringsbufferten justeras till 8,0 efter tillsats av reducerat glutation vid r...

<table border="1"><tbody><tr><td> Namnet på reagens </td><td> Företaget </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> pGEX4T-1-vektorn </td><td> GE Healthcare </td><td> 28-9545-49 </td><td> tidigare Amersham Biosciences </td></tr><tr><td> pMAL-c2-vektorn </td><td> New England BioLabs </td><td></td><td></td></tr><tr><td> pET30 vektor </td><td> EMD Chemicals </td><td> 69.077-3 </td><td> tidigare Novagen </td></tr><tr><td> Glutation-agarospärlor </td><td> BD Biosciences </td><td> 554.780 </td><td></td></tr><tr><td> Amylosharts </td><td> New England BioLabs </td><td> E8021S </td><td></td></tr><tr><td> Talon pärlor </td><td> Clontech </td><td> 635.501 </td><td></td></tr><tr><td> reducerad glutation </td><td> BD Biosciences </td><td> 554.782 </td><td></td&gt; </tr><tr><td> imidazol </td><td> Fisher </td><td> BP305-50 </td><td></td></tr><tr><td> maltos </td><td> Fisher </td><td> BP684-500 </td><td></td></tr><tr><td> S-proteinet agaros </td><td> EMD Chemicals </td><td> 69.704-3 </td><td> tidigare Novagen </td></tr><tr><td> Anti-Flag HRP </td><td> Sigma </td><td> A8592 </td><td></td></tr><tr><td> Anti-CFTR IgG </td><td> Skräddarsydd </td><td> R1104 </td><td> MAb igenkännande CFTR epitop vid aa 722-734 </td></tr><tr><td> Anti-MRP2 IgG </td><td> Chemicon International </td><td> MAB4148 </td><td> Nu en del av Millipore </td></tr></tbody></table> Tabell 2. Specifika reagens och utrustning.

in vitro analysis of pdz-dependent cftr macromolecular signaling complexes

Cystisk fibros transmembran konduktans (CFTR), en kloridkanal belägen främst på de apikala membranen i epitelceller, spelar en avgörande roll i transepitelial vätska homeostas 1-3. CFTR har varit inblandad i två stora sjukdomar: cystisk fibros (CF) 4 och sekretoriska diarré 5. I CF är syntes eller funktionella aktiviteten av CFTR Cl-kanalen minskas. Denna störning drabbar cirka 1 på 2.500 vita i USA 6. Överdriven CFTR aktivitet har också varit inblandad i fall av toxin-inducerad sekretoriska diarré (t.ex. genom kolera toxin och värme stabilt E. coli enterotoxin) som stimulerar cAMP eller cGMP produktion i tarmen 7. Ackumulerande bevis tyder på förekomsten av fysiska och funktionella interaktioner mellan CFTR och ett växande antal andra proteiner, inklusive transportörer, jonkanaler, receptorer, kinaser, fosfataser, signalIng-molekyler, och cytoskelettala element och dessa interaktioner mellan CFTR och dess bindande proteiner har visat sig vara kritiskt involverade i regleringen av CFTR-medierad transepitelial jontransport in vitro och även in vivo 8-19. I detta protokoll, fokuserar vi bara på de metoder som stöd i studiet av samspelet mellan CFTR karboxylterminala svans, som har en protein-bindande motiv [kallas PSD95/Dlg1/ZO-1 (PDZ) motiv] och en grupp byggnadsställning proteiner, vilka innehåller en specifik bindningspartner modul kallad PDZ domäner. Hittills har flera olika PDZ ställningsdelar proteiner har rapporterats binda till den karboxylterminala änden av CFTR med olika affiniteter, såsom NHERF1 och NHERF2 och PDZK1 och PDZK2, CAL (CFTR-associerad ligand), Shank2, och gripa 20-27. Den PDZ motivet inom CFTR som är erkänd av PDZ ställning proteiner är de sista fyra aminosyrorna vid C-terminalen (dvs. 1477-DTRL-1480 i mänsklig CFTR) 20. Intressant nogCFTR kan binda mer än en PDZ-domänen av både NHERFs och PDZK1, om än med varierande affinitet 22. Detta multivalens med avseende på CFTR bindning har visat sig vara av funktionell betydelse, vilket antyder att PDZ ställningsdelar proteiner kan underlätta bildning av CFTR makromolekylär signalering komplex för specifika / selektiva och effektiva signalering i celler 16-18. Flera biokemiska analyser har utvecklats för att studera CFTR-involvera proteininteraktioner, såsom sam-immunoutfällning, neddragningstransistor analys parvisa bindningsanalys, kolorimetrisk parvisa bindningsanalys och makromolekylärt komplex sammansättning analys 16-19,28,29 . Här fokuserar vi på de detaljerade förfaranden för montering en PDZ motiv-beroende CFTR-innehållande makromolekylära komplex in vitro, som används i stor utsträckning av vårt laboratorium för att studera protein-protein eller domän-domän interaktioner mellan CFTR 16-19,28,29.

Watch this Scientific Journal Video about In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes at JoVE.com

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystisk fibros transmembran konduktans-regulator (CFTR), en epitelial kloridkanal, har rapporterats interagera med olika proteiner och reglera viktiga cellulära processer, däribland de CFTR PDZ-motivet-medierade interaktioner har dokumenterats väl. Detta protokoll beskriver metoder vi utvecklat för att montera en PDZ-beroende CFTR makromolekylära signalering komplex<em&gt; In vitro</em&gt;.

In vitro analys av PDZ-beroende Signaling CFTR makromolekylära komplex

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

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In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

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