Name: mitochondria-associated er membranes (mams) and glycosphingolipid enriched microdomains (gems): isolation from mouse brain
Uploaded: 2013-03-04T12:15:00
Description: Watch this Scientific Journal Video about Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain at JoVE.com

Vi anerkender det bidrag, Renata Sano i udformningen den oprindelige protokol. Ad&#39;A. holder Jewelers for børn (JFC) Begavet Chair i Genetik og genterapi. Dette arbejde blev finansieret delvist af NIH tilskud GM60905, DK52025 og CA021764, og de amerikanske libanesiske syriske associerede Charities (ALSAC).

<html> Følgende protokol er beregnet til isolering og oprensning af Mams og perler fra musehjerne Løsninger der kræves for Isolering af mitokondrier, Mams og perler Fraktionering til opnåelse af rå mitochondrial præparat: Opløsning A: 0,32 M saccharose, 1 mM NaHCO3, 1 mM MgCl2, 0,5 mM CaCl2 + proteasehæmmere (tilføj ferske efter behov) Opløsni...

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Stederne for kontakt mellem intracellulære membraner eller mellem organeller og plasmamembran celler repræsenterer dynamiske signalering platforme for grundlæggende cellulære processer. Den nøjagtige karakterisering af deres funktion og sammensætning under både fysiologiske og patologiske tilstande kræver pålidelige og reproducerbare oprensningsprotokoller. Fremgangsmåderne beskrevet her, er blevet specifikt optimeret ved vores laboratorium til isolering og oprensning af Mams og tilsvarende perler fra voksen m...

<table border="1"><tbody><tr><td> Navn på reagens / Material </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td></td><td></td><td></td><td> REAGENSER </td></tr><tr><td></td><td></td><td></td><td> Fraktionering </td></tr><tr><td> Saccharose </td><td> Fisher Scientific </td><td> S5-500 </td><td></td></tr><tr><td> Natriumbikarbonat </td><td> Sigma-Aldrich </td><td> S-5761 </td><td></td></tr><tr><td> Magnesium Chloride, Hexahydrat </td><td> Fisher Scientific </td><td> BP214-500 </td><td></td></tr><tr><td> Calciumchlorid, dihydrat </td><td> Sigma-Aldrich </td><td> C-5080 </td><td></td></tr><tr><td></td><td></td><td></td><td> MAM </td></tr><tr><td> D-Mannitol </td><td> Sigma-Aldrich </td><td> M9546-250G </td><td></td></tr><tr><td> Hepes </td><td> Fisher Scientific </td><td> BP310-500 </td><td></td></tr><tr><td> EGTA </td><td> Sigma-Aldrich </td><td> E4378-250G </td><td></td></tr><tr><td> BSA, fraktion V, Heat Shock, lyofilisat </td><td> Roche </td><td> 03-116-964-001 </td><td></td></tr><tr><td> Percoll </td><td> GE </td><td> 17-0891-02 </td><td></td></tr><tr><td></td><td></td><td></td><td> GEM </td></tr><tr><td> Triton X-100 </td><td> Sigma-Aldrich </td><td> T9284-500 ml </td><td></td></tr><tr><td> Sodium Chloride </td><td> Fisher Scientific </td><td> S271-3 </td><td></td></tr><tr><td> Tris-base </td><td> Roche </td><td> 03-118-142-001 </td><td></td></tr><tr><td> HCI </td><td> Fisher Scientific </td><td> A144S-500 </td><td></td></tr&gt; <tr><td> EDTA </td><td> Fisher Scientific </td><td> BP120-500 </td><td></td></tr><tr><td> Natriumdodecylsulfat (SDS) </td><td> Fisher Scientific </td><td> BP166-500 </td><td></td></tr><tr><td></td><td></td><td></td><td> Fælles </td></tr><tr><td> Proteaseinhibitorer Tabletter, Complete EDTA-free </td><td> Roche </td><td> 11-873-580-001 </td><td></td></tr><tr><td></td><td></td><td></td><td> UDSTYR </td></tr><tr><td> 2 ml Douce All-glasvæv Grinders </td><td> Kimble Chase </td><td> 885300-0002 </td><td></td></tr><tr><td> 15 ml polypropylen koniske centrifugerør, BD Falcon </td><td> BD </td><td> 352.097 </td><td></td></tr><tr><td> 30 ml rundbundet Glas centrifugerør </td><td> Kimble Chase </td><td> 45.500-30 </td><td></td></tr><tr><td> 15 ml rundbundet Glass CentriFuge Tubes </td><td> Kimble Chase </td><td> 45.500-15 </td><td></td></tr><tr><td> Ultracentrifuge rør, Ultra-Klare, Thinwall, 14x89 mm </td><td> Beckman-Coulter </td><td> 344.059 </td><td></td></tr><tr><td> Parafilm </td><td> Cole-Parmer </td><td> PM996 </td><td></td></tr><tr><td> Engangs Borosilicate Glass Pasteur pipetter, 9 &quot; </td><td> Fisher Scientific </td><td> 13 til 678-20C </td><td></td></tr></tbody></table>

mitochondria-associated er membranes (mams) and glycosphingolipid enriched microdomains (gems): isolation from mouse brain

Intracellulære organeller er yderst dynamiske strukturer med varierende form og sammensætning, som underkastes cellespecifikke indre og ydre signaler. Deres membraner er ofte sammenstillet med definerede kontakt sites, der bliver knudepunkter for udveksling af signalmolekyler og membran komponenter 1,2,3,4. De indbyrdes organellar membran mikrodomæner, der dannes mellem det endoplasmatiske reticulum (ER) og mitokondrier ved åbningen af IP3-sensitive Ca2 +-kanal er kendt som mitokondrier associeret-ER membraner eller Mams 4,5,6. Protein / lipid-sammensætning og biokemiske egenskaber af disse membranmaterialer kontaktsteder er blevet omfattende undersøgt særligt med hensyn til deres rolle i regulering af intracellulær Ca2 + 4,5,6. ER fungerer som det primære lager af intracellulære Ca2 +, og i denne egenskab regulerer et utal af cellulære processer nedstrøms af Ca2 +-signalering, including posttranslationel proteinfoldning og protein maturation7. Mitokondrier, på den anden side bibeholdes Ca2 +-homeostase ved buffering cytosolisk Ca2 +-koncentration og derved forhindrer iværksættelsen af apoptotiske veje nedstrøms for Ca2 + ubalance 4,8. Den dynamiske karakter af Mams gør dem ideelle steder at dissekere de grundlæggende cellulære mekanismer, herunder Ca 2 + signalering og regulering af mitokondrie Ca 2 + koncentration, lipidbiosyntese og transport, energi metabolisme og celleoverlevelse 4,9,10,11,12. Adskillige protokoller er blevet beskrevet til oprensning af disse mikrodomæner fra levervæv og dyrkede celler 13,14. Idet tidligere publicerede metoder i betragtning, har vi tilpasset en protokol til isolering af mitokondrier og Mams fra voksen musehjerne. Til denne procedure har vi tilføjet en ekstra oprensningstrin, nemlig en Triton X100 udvinding, som enables isolationen af ​​de glycosphingolipid beriget microdomain (GEM) fraktion af Mams. Disse GEM præparater deler flere proteinkomponenter med caveolae og lipidklumper, afledt fra plasmamembranen eller andre intracellulære membraner, og foreslås til at fungere som samle point for ophobning af receptorproteiner og for protein-protein interaktioner 4,15.

Baseret på vores erfaringer med at bruge denne protokol vi kan roligt anbefale det til isolering og oprensning af Mams, ædelstene og mitokondrie fraktioner fra musehjerne. Fremgangsmåden beskrevet er særdeles reproducerbar og konsistent. I figur 1 viser vi et repræsentativt billede af den måde rent mitokondrier og Mams lag på en Percoll-gradient (trin 3.4). En defineret, mælkeagtig bånd indeholdende oprenset mitokondrier (Mito-P) segregerer i bunden af ​​ultracentrifugerør, mens MAM frakti...

Watch this Scientific Journal Video about Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain at JoVE.com

Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

Denne fremgangsmåde viser, hvordan at isolere fra den voksne musehjerne mitokondrierne-associerede ER membraner eller Mams og glycosphingolipid-berigede microdomain fraktioner fra Mams og mitokondriske præparater.

Mitokondrier-associerede ER Membranes (Mams) og glycosphingolipid Berigede mikrodomæner (GEM&#39;er): Isolering fra musehjerne

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic ...

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Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

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