Name: detection of protein interactions in plant using a gateway compatible bimolecular fluorescence complementation (bifc) system
Uploaded: 2011-09-16T13:00:00
Description: Watch this Scientific Journal Video about Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System at JoVE.com

Vi tackar Vi Nguyen för att läsa manuskriptet och allmän lab hjälp.

1. Beredning av Agrobacterium kultur <ol><li> Agrobacterium stam GV3101 förvandlas tidigare med Gateway kompatibel BiFC vektor pEarleyGate201-YC och pEarleyGate202-yn, var och en innehåller en blandning konstruktion av Indiens myndigheter. </li><li> Inokulera en 5-ml Yeb (5g L -1 Nötkött extrakt, 1g L -1 jästextrakt, 5g L -1 Peptone, 5g L -1 Sackaros, 2mm MgSO 4, pH 7,2) kultur BiFC fusionsprotein konstruerar förvandlas Agrobacterium</e...

<ol>
	<li>Ohad, N., Yalovsky, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilizing+bimolecular+fluorescence+complementation+(BiFC)+to+assay+protein-protein+interaction+in+plants.">Utilizing bimolecular fluorescence complementation (BiFC) to assay protein-protein interaction in plants.</a> Methods in molecular biology. 655, 347-358 (2010).</li><li>Fang, Y., Spector, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BiFC+imaging+assay+for+plant+protein-protein+interactions.">BiFC imaging assay for plant protein-protein interactions.</a> Cold Spring Harbor protocols. 2, (2010).</li><li>Schutze, K., Harter, K., Chaban, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimolecular+fluorescence+complementation+(BiFC)+to+study+protein-protein+interactions+in+living+plant+cells.">Bimolecular fluorescence complementation (BiFC) to study protein-protein interactions in living plant cells.</a> Methods in molecular biology. 479, 189-202 (2009).</li><li>Hiatt, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimolecular+fluorescence+complementation+(BiFC)+analysis+of+protein+interactions+in+Caenorhabditis+elegans.">Bimolecular fluorescence complementation (BiFC) analysis of protein interactions in Caenorhabditis elegans.</a> Methods. 45, 185-191 (2008).</li><li>Barnard, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+implementation+of+split-GFP-based+bimolecular+fluorescence+complementation+(BiFC)+assays+in+yeast.">Development and implementation of split-GFP-based bimolecular fluorescence complementation (BiFC) assays in yeast.</a> Biochemical Society Transactions. 36, 479-482 (2008).</li><li>Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+implementation+of+bimolecular+fluorescence+complementation+(BiFC)+assays+for+the+visualization+of+protein+interactions+in+living+cells.">Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells.</a> Nature. 1, 1278-1286 (2006).</li><li>Hu, C. D., Grinberg, A. V., Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+protein+interactions+in+living+cells+using+bimolecular+fluorescence+complementation+(BiFC)+analysis.">Visualization of protein interactions in living cells using bimolecular fluorescence complementation (BiFC) analysis.</a> Current protocols in cell biology. 21, 3-3 (2006).</li><li>Earley, K. 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BiFC-analysen är ett kraftfullt verktyg för att studera protein interaktioner. Till skillnad från den traditionella Y2H analysen BiFC inte bara tillåter visualisering av protein-protein växelverkan, men ger också mer information om proteinkomplex som sub-cellulära lokalisering. Dessutom är det möjligt att upptäcka indirekt interaktion så länge som de två kandidaten proteiner kan föras tillräckligt nära med en tredje partner. Precis som alla teknik, har BiFC sina begränsningar. På grund av kravet på mo...

<table border='1'>
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Acetosyringone</td>
			<td>Sigma</td>
			<td>D134406</td>
		</tr>
		<tr>
			<td>MES hydrate</td>
			<td>Sigma</td>
			<td>M2933</td>
		</tr>
		<tr>
			<td>1 mL slip-tip tuberculin syringe</td>
			<td>BD</td>
			<td>309602</td>
		</tr>
	</tbody>
</table>

detection of protein interactions in plant using a gateway compatible bimolecular fluorescence complementation (bifc) system

Vi har utvecklat en BiFC teknik för att testa samspelet mellan två proteiner in vivo. Detta sker genom att dela en gul fluorescerande protein (YFP) i två icke överlappande fragment. Varje fragment är klonade i-ram till en gen av intresse. Dessa konstruktioner kan sedan samarbeta omvandlas till Nicotiana benthamiana via Agrobacterium medierad omvandling, tillåter transitering uttryck för fusionsproteiner. Blandningen av YFP signalen bara uppstår när förhöret proteiner interagerar 1-7. Att testa och validera den protein-protein interaktioner kan BiFC användas tillsammans med jäst två hybrid (Y2H) analys. Detta kan detektera indirekt interaktion som kan förbises i Y2H. Gateway-tekniken är en universell plattform som gör det möjligt för forskare att skytteltrafik genen av intresse (GOI) i lika många uttryck och funktionella system analys som möjligt 8,9. Både orienteringen och läsram kan upprätthållas utan att använda restriktionsenzymer eller ligatur för att göra uttryck redo kloner. Som ett resultat kan man eliminera alla re-sekvensering åtgärder för att säkerställa enhetliga resultat i hela experimenten. Vi har skapat en serie gateway-kompatibel BiFC och Y2H vektorer som ger forskare med enkel att använda verktyg för att utföra både BiFC och Y2H analyser 10. Här visar vi hur lätt använda vår BiFC system för att testa protein-protein interaktioner i N. benthamiana växter.

Watch this Scientific Journal Video about Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System at JoVE.com

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

Vi har utvecklat en teknik för att testa protein-protein interaktioner i anläggningen. En gul fluorescerande protein (YFP) delas upp i två icke-överlappande fragment. Varje fragment är klonade i-ram till en gen av intresse via gateway, vilket gör att uttryck av fusionsproteiner. Ombildning av YFP signalen bara uppstår när förhöret proteiner interagerar.

Detektion av protein interaktioner i Plant använder gateway Kompatibel komplettering Bimolecular Fluorescence (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

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Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

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