Name: metabolic labeling and membrane fractionation for comparative proteomic analysis of arabidopsis thaliana suspension cell cultures
Uploaded: 2013-09-28T13:00:00
Description: Watch this Scientific Journal Video about Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures at JoVE.com

FREMGANGSMÅDE Fælles reagenser og buffere, der anvendes i udvinding protokol: <ol><li> JPL medium for Arabidopsis thaliana suspension cellekulturer <ul><li> 3 uM H 3 BO 3 </li><li> 3 uM MnSO4 x H 2 O </li><li> 1,1 uM ZnSO4 x 7 H2O </li><li> 0,15 uM KJ </li><li> 0,03 uM Na 2 MoO 4 x 2 H2O </li><li> 3 nM CoCl2 x 6 H2O </li><li> 3 nM CuSO4 x 5 H2O<...

<ol>
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Protokollen præsenteres i dette papir indeholder mange trin, og alle af dem er afgørende for at opnå ren og repræsentativ fraktionering af anlægget plasmamembranen i vaskemidler resistente membraner og detergentopløselige fraktioner. Derfor er det vigtigt at følge hvert trin som anvist. Behandling af membranen plasmafraktionen med ikke-ionisk detergent (trin 3.2) har den stærkeste indflydelse på kvaliteten af ​​membranen microdomain fraktionering. For at opnå reproducerbare res...

I 1972 Jonathan Singer og Garth Nicolson foreslog flydende mosaik model en struktur model af cellulære membraner, erstatter den protein-lipid-protein-sandwich model, der blev generelt accepteret i begyndelsen af ​​1960&#39;erne. Sanger og Nicolson postuleres, at den biologiske membran kan betragtes som en todimensionel væske, hvor alle lipid-og proteinmolekyler diffunderer frit og let 5. Siden dengang struktur model af plasmamembranen og kendskab til membranens sammensætning blev endnu mere kompleks. Især inden plasmamembranen strukturer såsom protein komplekser og lipid / sterol baseret strukturelt uordnede mikrodomæner kan iagttages. I kunstige modelmembraner 6,7, kan steroler og sphingolipider sideværts adskille fra andre lipidarter at danne områder med ændrede fysiske funktioner. Denne adskillelse inden for den cellulære membran er primært forårsaget af de selvstændige knytte egenskaber mellem steroler og meget saumættede kulbrinte kæder af phopsho-og sphingolipider 8. Især de stive sterol ringe favorisere interaktioner med stivere og mere lige mættede lipidarter og disse interaktioner tvinge tilstødende kulbrintekaeder i flere udvidede konformationer, øge membran tykkelse og hårdhed. En af de almindeligt observerede træk beriget membran mikrodomæner var deres uopløselighed ved behandling med non-ioniske detergenter, såsom Triton X-100 eller Brij 35. Disse fraktioner blev anset for at være identisk med membran mikrodomæner og blev kaldt vaskemiddel resistente membraner (DRM) på grundlag af deres biokemiske forberedelse metode 2. Brugen af ikke-ioniske detergenter under DRM udvinding modtaget nogle kritik som den biokemiske DRM præparatet kan ikke direkte svarer til nogen specifik membran rum inden for den levende celle 9. Især vaskemiddel til protein-forhold synes afgørende i sådanne præparater, ens forskellige rengøringsmidler, samt forskellige rengøringsmidler mængder kan give forskellig sammensætning af vaskemiddel modstandsdygtig membran fraktion 10. Der er imidlertid bevis for, at bestemte proteinarter specifikt forbinder med disse cellulære sterol-rige membran domæner, og at disse proteiner er godt beriget i biokemiske præparater af detergent-resistente membranfraktioner 11. Kernen af ​​proteiner, der blev fundet i plante DRM fraktion, og hvor tilstedeværelsen i DRM var sterol afhængige, var især GPI-forankrede proteiner, såsom fasciclin-lignende arabinogalactan proteiner (fla) og medlemmer af SKU protein familien. Også nogle signalproteiner, såsom receptor-lignende kinaser eller phospholipaser blev fundet 11. Disse resultater er i overensstemmelse med mange proteom undersøgelser om pattedyr membran mikrodomæner 12,13. Også i planter der er stigende evidens for den rolle membran mikrodomæner i forbindelse med stress respons 14 -16.. Den her beskrevne protokol giver en robust metode til fraktionering af plasma membran mikrodomæner og især benytter et protein til vaskemiddel koncentration, der giver os mulighed for at skildre stress inducerede ændringer af sterol-rige membran rum 4,11,14.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS </td>
		</tr>
		<tr>
			<td>Chemicals were ordered from Sigma-Aldrich unless noted otherwise</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ammonia (stock 25 % solution)</td>
			<td>WAKO</td>
			<td>010-03166</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TiO2 10 &mu;m</td>
			<td>GL-Science</td>
			<td>5020-75010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Empore Disk C18</td>
			<td>Varian</td>
			<td>12145004</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polyethylene glycol(PEG 3350)</td>
			<td>Sigma</td>
			<td>88276</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dextran T500</td>
			<td>Roth</td>
			<td>9219.2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Promega</td>
			<td>V5113</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail (PIC)</td>
			<td>Sigma</td>
			<td>P9599</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>K15NO3</td>
			<td>Cambridge Isotope Laboratories</td>
			<td>NLM-765-PK</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT </td>
		</tr>
		<tr>
			<td>Optima L-80 XP Ultracentrifuge</td>
			<td>Beckman</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>BioTek</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EASY-nLC II nano-Liquid Chromatograph</td>
			<td>Thermo Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LTQ-Orbitrap mass spectrometer</td>
			<td>Thermo Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifuge 5810R</td>
			<td>Eppendorf</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifuge 5417R</td>
			<td>Eppendorf</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Eppendorf</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Speed Vac RVC 2-25</td>
			<td>Christ</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Shaker Unimax 2010</td>
			<td>Heidolph</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

metabolic labeling and membrane fractionation for comparative proteomic analysis of arabidopsis thaliana suspension cell cultures

Plasmamembran mikrodomæner er funktioner baseret på de fysiske egenskaber af lipid-og sterol-miljø og har særlige roller i signalering processer. Udpakning beriget membran mikrodomæner fra planteceller for proteom analyse er en vanskelig opgave primært på grund af flere forberedelse trin og kilder for forureninger fra andre cellulære rum. Plasmamembranen kun udgør omkring 5-20% af alle membranerne i en plantecelle, og dermed isolering af fraktion højt oprenset plasmamembranen er udfordrende. En hyppigt anvendt metode involverer vandig to-faset opdeling i polyethylenglycol og dextran, som giver plasmamembranvesikler med en renhed på 95% 1. Sterol-rige membran mikrodomæner i plasmamembranen er uopløselige ved behandling med koldt ikke-ioniske detergenter ved alkalisk pH. Denne membranfraktion detergent-resistente kan separeres fra størstedelen plasmamembranen ved ultracentrifugering i såucrose gradient 2. Efterfølgende kan proteiner ekstraheres fra lav densitet bånd af saccharosegradienten med methanol / chloroform udfældning. Ekstraheret protein vil derefter blive trypsin fordøjet, afsaltet og endelig analyseret ved LC-MS/MS. Vores udvinding protokol for sterol-rige mikrodomæner er optimeret til fremstilling af rene detergent-resistente membranfraktioner fra Arabidopsis thaliana cellekulturer. Vi bruger fuld metabolisk mærkning af Arabidopsis thaliana suspension cellekulturer med K 15 NO 3 som den eneste kvælstof kilde til kvantitative komparative proteom undersøgelser efter biologisk behandling af interesse 3. Ved blanding af lige forhold af mærkede og umærkede cellekulturer for fælles proteinekstraktion indflydelse forberedelsestrin om den endelige kvantitativt resultat holdes på et minimum. Også tab af materiale under udvinding vil påvirke både kontrol og behandling prøver på samme måde, end derfor forholdet mellem lys og hævning peptid forbliver konstant. I den foreslåede metode enten mærket eller umærket cellekultur underkastes en biologisk behandling, mens den anden tjener som kontrol 4..

Med den præsenterede protokol hjælp metabolisk mærkede Arabidopsis cellekulturer er det muligt at isolere plasmamembraner fra plantevæv (trin 2, figur 2 og 4), og berige for vaskemiddel resistent membranfraktioner i plasmamembranen (trin 3, figur 3 og 5). Efterfølgende protokollen tillader udvinding af proteiner fra disse vaskemidler resistent membranfraktioner (trin 4) og fordøjelse af proteinet til sammenlignende proteom analyse (trin 5). Endelig valgfri berigelse af ph...

Watch this Scientific Journal Video about Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures at JoVE.com

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Her beskriver vi en robust fremgangsmåde til fraktionering af plante-plasmamembraner i detergent-resistente og detergentopløselige membraner baseret på en blanding af umærket og in vivo fuldt 15 N mærkede Arabidopsis thaliana cellekulturer. Proceduren anvendes til komparative proteom undersøgelser for at forstå signalering processer.

Metabolisk mærkning og membranfraktionering for Komparativ proteomiske analyse af<em&gt; Arabidopsis thaliana</em&gt; Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

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Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

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