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

PROSEDYRE Vanlige reagenser og buffere som brukes i ekstraksjonen protokoll: <ol><li> JPL medium for Arabidopsis thaliana suspensjon cellekulturer <ul><li> 3 mikrometer H 3 BO 3 </li><li> 3 mikrometer MnSO 4 x H 2 O </li><li> 1,1 mikrometer ZnSO 4 x 7 H 2 O </li><li> 0,15 mikrometer KJ </li><li> 0,03 mikrometer Na 2 Moo 4 x 2 H 2 O </li><li> 3 nM CoCl 2 x 6 H 2 O...

<ol>
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Protokollen som presenteres i denne artikkelen inneholder mange trinn og alle av dem er helt avgjørende for å oppnå ren og representative fraksjonering av planteplasma membran inn vaskemiddelresistente membraner og detergentløselige fraksjoner. Derfor er det viktig å følge hvert trinn som instruert. Behandling av plasmamembranfraksjonen med ikke-ionisk detergent (trinn 3.2) har størst innvirkning på kvaliteten av membranen microdomain fraksjonering. For å oppnå reproduserbare resul...

I 1972, Jonathan Singer og Garth Nicolson foreslo væske mosaikk-modellen en struktur modell av cellemembraner, erstatte protein-lipid-protein sandwich-modellen som var allment akseptert i de tidlige 1960-tallet. Singer og Nicolson postulerte at den biologiske hinne kan betraktes som en to-dimensjonal væske der alle lipid-og proteinmolekyler diffundere fritt og lett 5. Siden den gang struktur modell av plasmamembranen og kunnskap om membransammensetning ble enda mer komplisert. Spesielt, i plasmamembranen, strukturer som proteinkomplekser og lipid / sterol basert strukturelt uordnede microdomains kan observeres. I kunstig modellmembraner 6,7, kan steroler og sphingolipids lateralt skille fra andre lipid arter å danne regioner med endrede fysiske egenskaper. Dette segregering innenfor cellemembranen er hovedsakelig forårsaket av de selv assosiere egenskaper mellom steroler og svært saumettede hydrokarbon kjeder av phopsho-og sphingolipids åtte. Spesielt de stive sterol ringer favorisere interaksjoner med stivere og rettere mettet lipid arter og disse interaksjonene tvinge nabohydrokarbonkjeder i flere utvidede konformasjoner, økende membrantykkelse og hardhet. En av de vanligst observerte egenskaper av sterol anriket membran microdomains var deres uoppløselighet ved behandling med ikke-ioniske detergenter, for eksempel en Triton X-100 eller Brij 35.. Disse fraksjonene ble antatt å være identisk med membran microdomains og ble kalt vaskemiddelbestandige membraner (DRM) basert på deres biokjemiske forberedelse metode to. Bruken av ikke-ioniske vaskemidler i DRM ekstraksjon fikk noe kritikk som den biokjemiske DRM preparatet kan ikke direkte tilsvare en hvilken som helst spesifikk membrankammeret i den levende celle 9.. Spesielt synes vaskemiddel til protein ratio avgjørende i slike preparater, ens forskjellige vaskemidler, samt forskjellige detergentmengder kan gi forskjellig sammensetning av vaskemiddelbestandig membran fraksjon 10.. Men det er bevis for at spesielle protein arter spesielt forbinder med disse cellulære sterol-rik membran domener, og at disse proteinene er godt beriket i biokjemiske preparater av vaskemiddel-resistent membran fraksjoner 11. Kjernen av proteiner som ble funnet i anlegget DRM brøkdel, og hvor tilstedeværelse i DRMS ​​var sterol avhengig, var spesielt GPI-forankrede proteiner, slik som fasciclin lignende arabinogalaktan proteiner (FLAS) og medlemmer av SKU protein familien. Også noen signaleringsproteiner, slik som reseptor-lignende kinaser eller fosfolipaser ble funnet 11.. Disse resultatene er i samsvar med mange proteomikk studier på pattedyr membran microdomains 12,13. Også i planter er det økende bevis for rollen av membran microdomains i sammenheng med stressrespons 14 -16. Protokollen er beskrevet her gir en robust metode for fraksjonering av plasma membran microdomains og særlig bruker et protein til vaskemiddel konsentrasjon som tillater oss å skildre stress-induserte forandringer av sterol rike membran rommet 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 microdomains er funksjoner basert på de fysiske egenskaper av lipid og sterol miljø og har spesielle roller i signaleringsprosesser. Trekke sterol-beriket membran microdomains fra planteceller for proteomikk analyser er en vanskelig oppgave i hovedsak på grunn av flere forberedende trinn og kilder for forurensing fra andre cellene. Den plasmamembranen utgjør bare omtrent 5 til 20% av alle membranene i en plantecelle, og således isolasjonen av høyt renset plasmamembranfraksjonen er utfordrende. En ofte brukt metode involverer vandig to-fasepartisjone i polyetylenglykol og dekstran, som gir plasma membran-vesikler med en renhet på 95% 1. Sterol-rik membran microdomains i plasmamembranen er uoppløselig ved behandling med kaldt ikke-ioniske tensider ved alkalisk pH. Dette vaskemiddel-resistente membranfraksjon kan separeres fra hoveddelen plasma membran ved ultrasentrifugering i såucrose gradient to. Deretter kan proteiner ekstraheres fra den lave tetthet bandet av sukrose gradient av metanol / kloroform utfelling. Ekstrahert protein blir deretter trypsin-spaltet, avsaltet og endelig analysert ved LC-MS/MS. Vår ekstraksjon protokoll for sterol-rike microdomains er optimalisert for fremstilling av rene vaskemiddelresistent membranfraksjoner fra Arabidopsis thaliana cellekulturer. Vi bruker hele metabolske merking av Arabidopsis thaliana suspensjon cellekulturer med K 15 NO 3 som eneste nitrogenkilden for kvantitative komparative proteomikk studier etter biologisk behandling av interesse tre. Ved å blande like forhold av merkede og umerkede cellekulturer for felles protein ekstraksjon påvirkning av forberedelse trinn for slutt kvantitative resultat holdes på et minimum. Også tap av materiale under utvinning vil påvirke både kontroll og prøver behandling på samme måte, end derfor forholdet mellom lys og heave peptid vil forbli konstant. I den foreslåtte fremgangsmåte, enten merket eller umerket cellekultur gjennomgår en biologisk behandling, mens den andre tjener som kontroll 4..

Med den fremlagte protokoll ved hjelp av metabolisk merkede Arabidopsis cellekulturer er det mulig å isolere plasmamembraner fra plantevev (trinn 2, 4 Figur 2 og), og berike for detergentresistent membranfraksjoner innenfor plasmamembranen (trinn 3, figur 3 og 5). Deretter lar protokollen utvinning av proteiner fra disse vaskemiddelresistent membranfraksjoner (trinn 4), og fordøyelse av proteinet for sammenlignende analyse proteomikk (trinn 5). Til slutt, er det valgfritt ber...

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 fremgangsmåte for fraksjonering av planteplasmamembraner i detergentresistente og detergentløselige membraner basert på en blanding av umerket og in vivo fullt 15 N merket Arabidopsis thaliana cellekulturer. Fremgangsmåten er brukt for sammenlignende proteomic studier for å forstå signaleringsprosesser.

Metabolsk Merking og Membran Fraksjone for komparativ proteomikk analyser av<em&gt; Arabidopsis thaliana</em&gt; Suspension cellekulturer

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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In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...