Name: membrane-spine: a biochemical tool to identify protein-protein interactions of membrane proteins in vivo
Uploaded: 2013-11-07T19:35:00
Description: Watch this Scientific Journal Video about Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo at JoVE.com

Denne forskning blev støttet af tilskud fra Deutsche Forschungsgemeinschaft GraKo1121, Hu1011/2-1 og SFB940 til SH

Bemærk: Detaljerede oplysninger om buffere er angivet i protokollen findes i tabel 1. 1.. Fiksering af protein-protein interaktioner med formaldehyd Cross-sammenkædning i levende celler <ol><li> Til at begynde at forberede vækst medier, stamopløsninger og buffer P1 <ol><li> Forbered 500 ml medium: Mediet bør give vilkår, som understøtter samspillet mellem membranen bait protein og prey-proteiner. Desuden bør et eksperiment udføres under betingelser, hvor der forve...

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Membran SPINE analyse er en biokemisk tilgang, der gør det muligt at bekræfte og identificere til dette punkt ukendte interaktion partnere membranproteiner. Membran SPINE kombinerer in vivo krydsbinding af formaldehyd med oprensning af en Strep-mærket membran bait protein. Kombinationen med immunoblotting letter bekræftelse af forudsagte interaktionspartnere (figur 2). Derudover kombination med MS-analyse tillader identifikation af ukendte interaktion partnere (figur 3). Fo...

For at forstå funktionen af ​​et protein, er det vigtigt at kende dens interaktionspartnere. Flere klassiske teknikker er til rådighed til identifikation af interaktions partnere opløselige proteiner. Men disse teknikker er ikke let overføres til membranproteiner på grund af deres hydrofobe natur 4. For at overvinde denne begrænsning, har vi udviklet Membrane Strep-protein interaktion eksperiment (Membrane-SPINE) 6.. Den er baseret på SPINE metode, som kun var egnet til opløselige proteiner 7. Membran-Spine fordelene ved to fordele ved den tværbindingsmiddel formaldehyd: For det første kan formaldehyd let trænge membraner, og derfor frembringer et præcist øjebliksbillede af interactome i en levende celle 8. For det andet, kan formaldehyd tværbindinger vendes ved kogning 9. Her er disse to fordele bruges til at identificere ikke kun permanente, men også forbigående producentprisindeks membran proteins 6. Kort fortalt er en Strep-mærke fusioneret til den C-terminale ende af integral membran agn protein. Celler, der udtrykker membran agn protein inkuberes med formaldehyd, som tværbindinger bytte proteiner til membranen agn protein (figur 1). Modifikationer af prey-proteiner er ikke nødvendig. Dernæst membranfraktionen er forberedt. Derfor er membranproteiner opløseliggøres ved detergentbehandling og agn proteiner co-oprenset med sine prey-proteiner ved hjælp af affinitetsoprensning. Efterfølgende tværbinding vendes ved kogning og agn og dens co-eluerede prey-proteiner adskilles ved SDS-PAGE. Endelig kan prey-proteiner identificeres ved immunoblotanalyse eller massespektrometri.

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			<td height="21" style="height:21px;width:343px;">37% Formaldehyde</td>
			<td style="width:128px;">Roth</td>
			<td style="width:137px;">4979.1</td>
			<td style="width:271px;">Should not be older than one year</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DNaseI</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:137px;">DN25</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">BCA protein assay kit</td>
			<td style="width:128px;">Pierce</td>
			<td style="width:137px;">23225</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Micromagnetic rod</td>
			<td style="width:128px;">Roth</td>
			<td style="width:137px;">0955.2</td>
			<td style="width:271px;">5 mm in length, 2 mm in diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Triton X-100</td>
			<td style="width:128px;">Roth</td>
			<td style="width:137px;">6683.1</td>
			<td style="width:271px;">standard detergent for solubilization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">n-Dodecyl-&#946;-maltoside</td>
			<td style="width:128px;">Glycon</td>
			<td style="width:137px;">D97002-C</td>
			<td style="width:271px;">the best detergent to solubilze CpxA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">1 ml Strep-Tactin superflow gravity flow column</td>
			<td style="width:128px;">IBA</td>
			<td style="width:137px;">2-1207-050</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Strep-tag protein purification buffer set</td>
			<td style="width:128px;">IBA</td>
			<td style="width:137px;">2-1002-001</td>
			<td style="width:271px;">Contains buffer W and buffer E</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Amicon Ultra-4 centrifugal filter</td>
			<td style="width:128px;">Millipore</td>
			<td style="width:137px;">UFC803024</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">SuperSignal West Pico Chemiluminescent substrate</td>
			<td style="width:128px;">Pierce</td>
			<td style="width:137px;">34080</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">SilverQuest Silverstaining kit</td>
			<td style="width:128px;">Invitrogen</td>
			<td style="width:137px;">LC6070</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">FireSilver Staining Kit</td>
			<td style="width:128px;">Proteome Factory</td>
			<td style="width:137px;">P-S-2001</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Ultracentrifuge</td>
			<td style="width:128px;"></td>
			<td style="width:137px;"></td>
			<td style="width:271px;"></td>
		</tr>
	</tbody>
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membrane-spine: a biochemical tool to identify protein-protein interactions of membrane proteins in vivo

Membranproteiner er essentielle for cellelevedygtighed, og er derfor vigtige terapeutiske mål 1-3. Da de fungerer i komplekser 4, er det nødvendigt 5 metoder til at identificere og karakterisere deres samspil. Til dette formål har vi udviklet Membrane Strep-protein-interaktion eksperiment, kaldet Membrane-SPINE 6. Denne teknik kombinerer vivo tværbinding ved hjælp af den reversible tværbinder formaldehyd med affinitetsoprensning af et Strep-mærket membran bait protein. Under proceduren tværbundne prey-proteiner co-oprenset med membranen bait-proteinet og efterfølgende separeret ved kogning. Derfor kan to store opgaver skal udføres, når man analyserer protein-protein interaktioner (PPI) af membranproteiner hjælp Membran-SPINE: første, bekræftelse af en foreslået interaktion partner ved immunoblotting, og for det andet, identifikation af nye interaktions partnere ved massespektrometrianalyse . Selv low affinitet forbigående PPI er påviselige ved denne teknik. Endelig Membrane-SPINE kan tilpasses til næsten enhver celletype, hvilket gør den anvendelig som et kraftfuldt screeningsværktøj til at identificere producentprisindeks for membranproteiner.

Membran SPINE analyse tillader co-oprensning af membranproteiner og transient interagerende protein partnere. Den co-oprensning opnås ved hjælp af cross-linking agent formaldehyd. To parametre er afgørende for at forhindre uspecifikke tværbindinger: formaldehydkoncentrationen og krydsbinding gang. Den tilstrækkelige, men ikke overdreven brug af formaldehyd kan let kontrolleres ved immunoblotting. Formaldehyd tværbundne protein-komplekser kan separeres ved kogning, men ikke ved SDS behandling. Derfor kan de visuali...

Watch this Scientific Journal Video about Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo at JoVE.com

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

En biokemisk fremgangsmåde er beskrevet til at identificere in vivo protein-protein interaktioner (PPI) af membranproteiner. Fremgangsmåden kombinerer protein tværbinding affinitetsoprensning og massespektrometri, og kan tilpasses til næsten enhver celletype eller organisme. Med denne fremgangsmåde bliver det muligt selv identifikation af forbigående PPI.

Membran-SPINE: en biokemisk redskab til at identificere protein-protein interaktioner membranproteiner<em&gt; In Vivo</em

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

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Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

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