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 Nguyen Biz el yazması ve genel laboratuvar yardım okuma için teşekkür ederim.

1. Agrobacterium kültür hazırlanması <ol><li> Agrobacterium suşu GV3101 önceden Ağ Geçidi uyumlu BiFC vektör pEarleyGate201-YC ve pEarleyGate202-YN, her bir füzyon GOI inşa içeren ile değiştirdi. </li><li> BiFC füzyon proteini bir 5 ml YEB (5g L -1 Sığır özü, 1g L -1 Maya özü, 5g L -1 Peptonlu, 5g L -1 Sakkaroz, 2mm MgSO 4, pH 7,2) kültür İnokülasyon ile Agrobacterium dönüştürülmüş yapıları uygun antibi...

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	<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. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gateway-compatible+vectors+for+plant+functional+genomics+and+proteomics.">Gateway-compatible vectors for plant functional genomics and proteomics.</a> The Plant journal : for cell and molecular biology. 45, 616-629 (2006).</li><li>Karimi, M., Inze, D., Depicker, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GATEWAY+vectors+for+Agrobacterium-mediated+plant+transformation.">GATEWAY vectors for Agrobacterium-mediated plant transformation.</a> Trends in Plant Science. 7, 193-195 (2002).</li><li>Lu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+homolog+of+the+yeast+TREX-2+mRNA+export+complex:+components+and+anchoring+nucleoporin.">Arabidopsis homolog of the yeast TREX-2 mRNA export complex: components and anchoring nucleoporin.</a> The Plant journal : for cell and molecular biology. 61, 259-270 (2010).</li><li>Sparkes, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=transient+expression+of+fluorescent+fusion+proteins+in+tobacco+plants+and+generation+of+stably+transformed+plants.">transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants.</a> Nature protocols. 1, 2019-2025 (2006).</li><li>Rackham, O., Brown, C. M. <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+RNA-protein+interactions+in+living+cells:+FMRP+and+IMP1+interact+on+mRNAs.">Visualization of RNA-protein interactions in living cells: FMRP and IMP1 interact on mRNAs.</a> The EMBO journal. 23, 3346-3355 (2004).</li><li>Demidov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+complementation+of+split+fluorescent+protein+triggered+by+DNA+hybridization.">Fast complementation of split fluorescent protein triggered by DNA hybridization.</a> Proceedings of the National Academy of Sciences of the United States of America. 103, 2052-2056 (2006).</li><li>Shyu, Y. J., Suarez, C. D., Hu, C. D. <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+ternary+complexes+in+living+cells+by+using+a+BiFC-based+FRET+assay.">Visualization of ternary complexes in living cells by using a BiFC-based FRET assay.</a> Nature. 3, 1693-1702 (2008).</li></ol>

BiFC tahlil protein etkileşimleri çalışmak için güçlü bir araçtır. BiFC geleneksel Y2H testinin aksine, sadece protein-protein etkileşimleri görselleştirme sağlar, ama aynı zamanda alt hücresel yerleşimi gibi protein kompleksi daha fazla bilgi sağlar. Ayrıca, iki aday proteinler üçüncü bir ortağı tarafından yeterince yakın getirilebilir olarak sürece dolaylı etkileşimleri tespit etmek mümkündür. Herhangi bir teknoloji gibi, BiFC sınırlamaları vardır. BiFC fluorofor oluşumu için mol...

<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

Biz, in vivo olarak iki protein arasındaki etkileşimi test etmek için bir BiFC tekniği geliştirdik . Bu örtüşmeyen iki parçalara bölme sarı floresan protein (YFP) tarafından gerçekleştirilir. Her parçası ilgi bir gen-frame klonlanmış. Bu yapılardan sonra, füzyon proteinleri transit ifade izin Agrobacterium aracılı dönüşüm yoluyla Nicotiana benthamiana içine ortak dönüştürülmüş olabilir. YFP sinyal sulandırıldıktan sadece tahkikat proteinleri 1-7 etkileşime girdiğinde oluşur. BiFC protein-protein etkileşimleri test etmek ve doğrulamak için, maya iki hibrid (Y2H) tayini ile birlikte kullanıldığında olabilir. Bu Y2H göz ardı edilebilir dolaylı etkileşimleri algılayabilir. Ağ Geçidi teknoloji, servis ilgi gen (GOI) araştırmacıları, evrensel bir platformda pek çok ifade ve mümkün 8,9 olarak fonksiyonel analiz sistemleri. Her iki yönelim ve okuma çerçevesini ifade hazır klonları yapmak için restriksiyon enzimleri veya ligasyonu kullanmadan muhafaza edilebilir. Sonuç olarak, bir deneyler boyunca tutarlı sonuçları elde etmek için yeniden sıralama adımları ortadan kaldırabilir. Biz, hem de BiFC ve Y2H testleri gerçekleştirmek için 10 kolay kullanımlı araçları ile araştırmacılara Ağ Geçidi uyumlu BiFC ve Y2H vektörler bir dizi oluşturduk . Burada, N. protein-protein etkileşimleri test etmek için BiFC sistemini kullanarak kolaylığı göstermek benthamiana bitkiler.

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

Biz bitki protein-protein etkileşimleri test etmek için bir teknik geliştirdik. Sarı floresan protein (YFP) örtüşmeyen iki parça halinde bölünmüş durumda. Her parçası füzyon proteinleri ifade sağlayan, Gateway sistemi üzerinden ilgi bir gen-frame klonlanmış. YFP sinyal Sulandırma sadece tahkikat proteinler etkileşime girdiğinde oluşur.

Ağ Geçidi Uyumlu Bimoleküler Floresan tamamlama (BiFC) kullanarak Bitki Protein Etkileşimleri Tespiti

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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Biology

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

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

Biyolojik Tahliller Manyetik Modülasyon Biyoalgılayıcı Sistemi kullanarak hızlı Homojen Algılama

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Helios Gen Tabancası kullanarak Soğan Hücrelerde Protein-Protein Etkileşimi Bimoleküler Floresan tamamlama (BiFC) Testi

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Mikro desenli Yüzeyler, protein-protein etkileşimleri-vivo Algılama

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Bimolecular Fluorescence Complementation

Bimoleküler Floresan tamamlama

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Dayanarak protein-protein etkileşimleri ve kimliği için bir protokol<sup&gt; 15</sup&gt; K Metabolik Etiketleme, immünopresipitasyon, Kantitatif Kütle Spektrometrisi ve Affinity Modülasyon

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Bimoleküler Floresan tamamlama akışı Sitometrik Analizi: Protein-protein etkileşim Eğitim Bir Yüksek Verimli Kantitatif Yöntemi

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Tütün protoplastlarında ve Yapraklar Bimoleküler Floresan tamamlama tarafından Visualized protein-protein etkileşimleri

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Biyoparlaklık Rezonans Enerji Transferi kullanma Canlı Hücrelerde Protein-protein etkileşimleri araştıran

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

Diferansiyel Taramalı florimetri Kullanarak Protein-ligand Etkileşimleri belirlenmesi

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...