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

PROCEDURE Gemeenschappelijke reagentia en buffers die in de extractie protocol: <ol><li> JPL medium voor Arabidopsis thaliana schorsing celculturen <ul><li> 3 uM H 3 BO 3 </li><li> 3 uM MnSO 4 x H2O </li><li> 1.1 uM ZnSO 4 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</s...

<ol>
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De in dit document protocol bevat vele stappen en ze zijn cruciaal voor zuivere en representatieve fractionering van de plant plasmamembraan in reinigingsmiddelenbestendige membranen en detergent oplosbare fracties te verkrijgen. Daarom is het belangrijk om elke stap te volgen instructies. Behandeling van de plasmamembraan fractie met niet-ionisch detergens (stap 3.2) heeft de sterkste invloed op de kwaliteit van het membraan microdomain fractionering. Om reproduceerbare resultaten te verkri...

In 1972, Jonathan Singer en Garth Nicolson voorgesteld de vloeistof mozaïek model een structuur model van cellulaire membranen, het vervangen van de eiwit-lipide-eiwit sandwich model dat in het algemeen in de vroege jaren 1960 werd aanvaard. Singer en Nicolson gepostuleerd dat het biologische membraan kan worden beschouwd als een tweedimensionale vloeistof waar alle lipiden en eiwitmoleculen vrij en gemakkelijk 5 diffunderen. Sindsdien structuurmodel van het plasmamembraan en kennis van het membraan preparaat werd nog ingewikkelder. Vooral in de plasmamembraan, structuren zoals eiwitcomplexen lipiden / sterol gebaseerd structureel verstoord microdomeinen worden waargenomen. In kunstmatige modelmembranen 6,7, kan sterolen en sphingolipiden zijdelings te scheiden van andere soorten lipiden aan regio&#39;s te vormen met gewijzigde fysieke kenmerken. Deze segregatie binnen het celmembraan wordt voornamelijk veroorzaakt door de zelfassocierende eigenschappen tussen sterolen en zeer saonverzadigde koolwaterstof ketens van phopsho-en sfingolipiden 8. Bijzonder, de starre sterol ringen gunst interacties met stijvere en rechtere verzadigde soorten lipiden en deze interacties dwingen naburige koolwaterstofketens in meer uitgebreide conformaties, toenemende membraandikte en hardheid. Een van de vaak waargenomen kenmerken sterol verrijkte membraan microdomeinen was hun onoplosbaarheid na behandeling met niet-ionische detergentia zoals Triton X-100 en Brij 35. Deze fracties werden verondersteld identiek met membraan microdomeinen te zijn en werden genoemd reinigingsmiddelenbestendige membranen (DRM) op basis van hun biochemische bereidingswijze 2. Het gebruik van niet-ionische detergenten tijdens DRM extractie kreeg kritiek omdat de biochemische DRM preparaat kan niet rechtstreeks overeen met een specifieke membraancompartiment binnen de levende cel 9. Vooral, het wasmiddel aan eiwitverhouding lijkt cruciaal in dergelijke preparaten, eenTussen verschillende detergenten, evenals verschillende hoeveelheden detergens kunnen andere samenstelling van de reinigingsmiddelenbestendige membraanfractie 10 opleveren. Er is bewijs dat bepaalde proteïne species specifiek associëren met deze cellulaire sterol-rijke membraan domeinen, en dat deze eiwitten zijn goed verrijkt biochemische preparaten wasmiddel-resistente membraan fracties 11. De kern van eiwitten die werden gevonden in plantaardige DRM fractie en waarbij de aanwezigheid DRM was sterol afhankelijk waren bijzonder GPI-verankerde eiwitten, zoals fascicline-achtige eiwitten arabinogalactaan (knip) en leden van de SKU eiwitfamilie. Ook enkele signalerende proteïnen, zoals receptor-achtige kinasen of fosfolipase gevonden 11. Deze resultaten zijn consistent met vele proteomische studies voor zoogdieren membraan microdomeinen 12,13. Ook in planten is er steeds meer bewijs voor de rol van membraan microdomeinen in context van stress respons 14 -16. De hier beschreven protocol voorziet in een robuuste methode voor fractionering van plasmamembraan microdomeinen en bijzonder maakt gebruik van een eiwit aan detergent concentratie die ons in staat stelt om stress geïnduceerde veranderingen van de sterol-rijke membraancompartiment 4,11,14 verbeelden.

<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

Plasmamembraan microdomeinen zijn functies op basis van de fysische eigenschappen van het lipide en sterol milieu en hebben een specifieke rol bij het signaleren van processen. Extraheren-sterol verrijkte membraan microdomeinen van plantencellen voor proteomics analyse is een moeilijke taak vooral te wijten aan meerdere voorbereidende stappen en de bronnen van verontreinigingen uit andere cellulaire compartimenten. Het plasmamembraan vormt slechts 5-20% van de membranen in een plantencel, en dus isolatie van sterk gezuiverde celmembraan fractie uitdagend. Een veel gebruikte methode bestaat waterige twee-fase verdeling in polyethyleenglycol en dextran, die plasmamembraan vesicles oplevert met een zuiverheid van 95% 1. Sterol-rijke membraan microdomeinen binnen het plasmamembraan onoplosbaar na behandeling met koud ionogene detergentia bij alkalische pH. Dit detergent-resistente membraan fractie kan vanuit de bulk plasmamembraan worden gescheiden door ultracentrifuge in alsucrose gradiënt 2. Vervolgens kunnen eiwitten worden geëxtraheerd uit de lage dichtheid band van de sucrose gradiënt van methanol / chloroform precipitatie. Geëxtraheerd eiwit vervolgens met trypsine worden gedigereerd, ontzout en tenslotte geanalyseerd met LC-MS/MS. De extractie protocol voor sterol-rijke microdomeinen is geoptimaliseerd voor de bereiding van schone detergens resistent membraan fracties uit Arabidopsis thaliana celkweken. We gebruiken volledige metabole kenmerken van Arabidopsis thaliana schorsing celculturen met K 15 NO 3 als de enige bron van stikstof voor kwantitatieve vergelijkende proteomics studies na biologische behandeling van belang 3. Door gelijke verhoudingen van gelabelde en ongelabelde celculturen gezamenlijke eiwitextractie invloed voorbereiding stappen uiteindelijke kwantitatief resultaat wordt minimaal gehouden. Ook verlies van materiaal tijdens de extractie zowel controle en behandeling monsters op dezelfde wijze affectd dus de verhouding van licht en bijdraaien peptide blijft constant. In de voorgestelde werkwijze ofwel gemerkt of ongemerkt celkweek ondergaat een biologische behandeling, terwijl de andere dient als controle 4.

Bij de gepresenteerde protocol met metabolisch gemerkt Arabidopsis celkweken kan men plasmamembranen isoleren van plantenweefsel (stap 2, figuren 2 en 4) en verrijken reinigingsmiddelenbestendige membraanfracties in het plasmamembraan (stap 3, figuren 3 en 5). Vervolgens het protocol maakt extractie van eiwitten uit deze reinigingsmiddelenbestendige membraanfracties (stap 4) en digestie van het eiwit vergelijkende proteoomanalyse (stap 5). Ten slotte is de optionele verrijking ...

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

Hier beschrijven we een robuuste werkwijze voor de fractionering van plantaardige plasmamembranen in reinigingsmiddelenbestendige en detergent oplosbare membranen gebaseerd op een mengsel van gelabelde en in vivo volledig 15N gemerkt Arabidopsis thaliana celculturen. De procedure wordt toegepast voor vergelijkende proteomics studies om signalering processen te begrijpen.

Metabole Labeling en Membrane Fractionation for Comparative Proteomic analyse van<em&gt; Arabidopsis thaliana</em&gt; Schorsing Celkweken

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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