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

Vores arbejde er blevet støttet af tilskud fra American Heart Association (Greater Sydøst Affiliate) Beginning-tilskud-in-støtte 0765185B, at Elsa U. Pardee Foundation forskningsbevilling, og Wayne State University murene opstart fond og Cardiovascular Research Institute Isis Initiative pris. Denne fremgangsmåde til in vitro CFTR makromolekylært kompleks samling blev oprindeligt udviklet af Dr. AP Naren (University of Tennessee Health Science Center).

1. Ekspression og oprensning af rekombinant mærkede fusionsproteiner i bakterier <ol><li> Amplificere definerede regioner i C-halerne (de sidste 50-100 aminosyrer indeholdende PDZ motiver ved C-terminus) af CFTR, LPA 2, MRP2, MRP4, β 2 AR og NHERFs (fuld længde eller pDZ1 eller PDZ2 domæner ) ved PCR-fremgangsmåde. </li><li> Klone PCR-produkterne ind i pGEX4T-1 vektor til GST-fusionsproteiner (såsom GST-NHERFs, GST-MRP4 CT), pMAL-c2 vektoren for MBP-fusionsproteiner (såsom MBP-β 2</s...

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I denne protokol vi demonstreret en fremgangsmåde til in vitro-samling og påvisning af en CFTR indeholdende makromolekylært signalering kompleks under anvendelse af oprensede proteiner (eller proteinfragmenter) og / eller cellelysater, som beskrevet tidligere 16-19,29,30. For at opnå det bedste resultat de følgende kritiske punkter under fremstillingsprocessen kræver særlig opmærksomhed: <ul><li> Det er vigtigt, at pH i elueringspufferen være justeret til 8,0 efter tilsætning af reducer...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nr. </td><td> Kommentarer </td></tr><tr><td> pGEX4T-1-vektor </td><td> GE Healthcare </td><td> 28-9545-49 </td><td> tidligere Amersham Biosciences </td></tr><tr><td> pMAL-c2 vektoren </td><td> New England BioLabs </td><td></td><td></td></tr><tr><td> pET30 vektor </td><td> EMD Kemikalier </td><td> 69077-3 </td><td> tidligere Novagen </td></tr><tr><td> Glutathionagaroseperler </td><td> BD Biosciences </td><td> 554780 </td><td></td></tr><tr><td> Amyloseharpiks </td><td> New England BioLabs </td><td> E8021S </td><td></td></tr><tr><td> Talon perler </td><td> Ciontech </td><td> 635501 </td><td></td></tr><tr><td> reduceret glutathion </td><td> BD Biosciences </td><td> 554782 </td><td></td&gt; </tr><tr><td> imidazol </td><td> Fisher </td><td> BP305-50 </td><td></td></tr><tr><td> maltose </td><td> Fisher </td><td> BP684-500 </td><td></td></tr><tr><td> S-protein agarose </td><td> EMD Kemikalier </td><td> 69704-3 </td><td> tidligere 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> Skræddersyet </td><td> R1104 </td><td> mAb, som genkender CFTR epitopen aa 722-734 </td></tr><tr><td> Anti-MRP2 IgG </td><td> Chemicon International </td><td> MAB4148 </td><td> Nu er en del af Millipore </td></tr></tbody></table> Tabel 2 nedenfor. Specifikke reagenser og udstyr.

in vitro analysis of pdz-dependent cftr macromolecular signaling complexes

Cystisk fibrose transmembran konduktans regulator (CFTR), en chlorid-kanal er placeret hovedsageligt på de apikale membraner i epitelceller, spiller en afgørende rolle i transepithelial fluid homøostase 1-3. CFTR har været impliceret i to vigtige sygdomme: cystisk fibrose (CF) 4 og sekretorisk diarré 5. I CF, er syntesen eller funktionelle aktivitet af CFTR Cl-kanal reduceret. Denne lidelse påvirker cirka 1 ud af 2.500 kaukasiere i USA 6. Overdreven CFTR-aktivitet har også været impliceret i tilfælde af toksin-induceret sekretorisk diarré (fx ved koleratoxin og varmestabil E. coli enterotoxin), der stimulerer cAMP eller cGMP-produktion i tarmen 7. Akkumulerende beviser antyder eksistensen af ​​fysiske og funktionelle interaktioner mellem CFTR og et stigende antal af andre proteiner, herunder transportører, ionkanaler, receptorer, kinaser, phosphataser, signalIng molekyler og cytoskeletale elementer, og disse interaktioner mellem CFTR og dets bindende proteiner har vist sig at være kritisk involveret i reguleringen af CFTR-medieret transepithelial iontransport in vitro og også in vivo 8-19. I denne protokol, fokuserer vi kun på de metoder, som støtte i studiet af samspillet mellem CFTR carboxylterminal hale, som besidder et protein-bindende motiv [benævnt PSD95/Dlg1/ZO-1 (PDZ) motiv], og en gruppe stillads proteiner, som indeholder en specifik bindingspartner modul benævnt PDZ-domæner. Hidtil har flere forskellige PDZ stilladsdele proteiner blevet rapporteret at binde til den carboxylterminale hale af CFTR med forskellige affiniteter, såsom NHERF1 og NHERF2 og PDZK1 og PDZK2 og CAL (CFTR-associeret ligand), Shank2, og gribe 20-27. PDZ motiv i CFTR, der genkendes af PDZ skelettet proteiner er de sidste fire aminosyrer ved C-terminus (dvs. 1477-DTRL-1480 i human CFTR) 20. InteressantCFTR kan binde mere end en PDZ-domæne af både NHERFs og PDZK1, selv med varierende affiniteter 22. Dette multivalens med hensyn til CFTR binding har vist sig at være funktionelle betydning, hvilket antyder, at PDZ stilladsdele proteiner kan lette dannelse af CFTR makromolekylære signalering komplekser til specifikke / selektive og effektive signalering i celler 16-18. Flere biokemiske assays er blevet udviklet til at studere CFTR-involverer protein-interaktioner, såsom co-immunopræcipitation, pull-down assay parvise bindingsassay, kolorimetrisk parvise bindingsassay, og makromolekylære komplekst assay 16-19,28,29 . Her fokuserer vi på de detaljerede procedurer for at samle et PDZ motiv-afhængig CFTR-holdige makromolekylære kompleks in vitro, som bruges i udstrakt grad af vores laboratorium til at studere protein-protein eller domæne-domæne-interaktioner, der involverer 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 fibrose transmembran konduktans regulator (CFTR), en epitelcelle chlorid-kanal, er blevet rapporteret at interagere med forskellige proteiner og regulere vigtige cellulære processer, deriblandt CFTR PDZ motiv-medierede interaktioner er blevet godt dokumenteret. Denne protokol beskriver de metoder vi har udviklet til at samle en PDZ-afhængig CFTR makromolekylære signalering kompleks<em&gt; In vitro</em&gt;.

In vitro-analyse af PDZ-afhængige CFTR Makromolekylære signalering Komplekser

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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