Name: protein membrane overlay assay: a protocol to test interaction between soluble and insoluble proteins in vitro
Uploaded: 2011-08-14T19:00:00
Description: Watch this Scientific Journal Video about Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro at JoVE.com

हमारी प्रयोगशाला में काम एनआईएच, खाद्य और कृषि USDA राष्ट्रीय संस्थान, NSF, चारण, डो, और बीएसएफ से कुलपति को अनुदान द्वारा समर्थित है

1. अभिव्यक्ति और प्रोटीन की निकासी <ol><li> विभिन्न उनके पता लगाने के लिए परीक्षण किया जा प्रोटीन टैग. लेबल प्रोटीन झिल्ली (ProIM) पर एक बड़े आकार (जैसे, जीएसटी) के एक टैग के साथ immobilized हो, और unfused टैग एक स्थिर नकारात्म?...

<ol>
	<li>Hu, C. D., Chinenov, Y., 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+interactions+among+bZIP+and+Rel+family+proteins+in+living+cells+using+bimolecular+fluorescence+complementation.">Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation.</a> Mol Cell. 9, 789-798 (2002).</li><li>Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+localization+of+interacting+proteins+by+bimolecular+fluorescence+complementation+in+planta.">Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta.</a> J Mol Biol. 362, 1120-1131 (2006).</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> Nat Protoc. 1, 1278-1286 (2006).</li><li>Shyu, Y. J. <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+Caenorhabditis+elegans+using+bimolecular+fluorescence+complementation+analysis.">Visualization of protein interactions in living Caenorhabditis elegans using bimolecular fluorescence complementation analysis.</a> Nat Protoc. 3, 588-596 (2008).</li><li>Citovsky, V., Gafni, Y., Tzfira, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localizing+protein-protein+interactions+by+bimolecular+fluorescence+complementation+in+planta.">Localizing protein-protein interactions by bimolecular fluorescence complementation in planta.</a> Methods. 45, 196-206 (2008).</li><li>Ohad, N., Shichrur, K., 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=The+analysis+of+protein-protein+interactions+in+plants+by+bimolecular+fluorescence+complementation.">The analysis of protein-protein interactions in plants by bimolecular fluorescence complementation.</a> Plant Physiol. 145, 1090-1099 (2007).</li><li>Zamyatnin, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+the+integral+membrane+protein+topology+in+living+cells.">Assessment of the integral membrane protein topology in living cells.</a> Plant J. 46, 145-154 (2006).</li><li>Citovsky, V., Knorr, D., Schuster, G., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+P30+movement+protein+of+tobacco+mosaic+virus+is+a+single-strand+nucleic+acid+binding+protein.">The P30 movement protein of tobacco mosaic virus is a single-strand nucleic acid binding protein.</a> Cell. 60, 637-647 (1990).</li><li>Heinlein, M., Epel, B. L., Padgett, H. S., Beachy, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+tobamovirus+movement+proteins+with+the+plant+cytoskeleton.">Interaction of tobamovirus movement proteins with the plant cytoskeleton.</a> Science. 270, 1983-1985 (1995).</li><li>Tomenius, K., Clapham, D., Meshi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+by+immunogold+cytochemistry+of+the+virus+coded+30+K+protein+in+plasmodesmata+of+leaves+infected+with+tobacco+mosaic+virus.">Localization by immunogold cytochemistry of the virus coded 30 K protein in plasmodesmata of leaves infected with tobacco mosaic virus.</a> Virology. 160, 363-371 (1987).</li><li>Ding, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+plasmodesmata+are+specific+sites+of+localization+of+the+tobacco+mosaic+virus+movement+protein+in+transgenic+tobacco+plants.">Secondary plasmodesmata are specific sites of localization of the tobacco mosaic virus movement protein in transgenic tobacco plants.</a> Plant Cell. 4, 915-928 (1992).</li><li>Meshi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+the+30+kd+protein+of+tobacco+mosaic+virus:+involvement+in+cell-to-cell+movement+and+dispensability+for+replication.">Function of the 30 kd protein of tobacco mosaic virus: involvement in cell-to-cell movement and dispensability for replication.</a> EMBO J. 6, 2557-2563 (1987).</li><li>Wolf, S., Deom, C. M., Beachy, R. N., Lucas, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Movement+protein+of+tobacco+mosaic+virus+modifies+plasmodesmatal+size+exclusion+limit.">Movement protein of tobacco mosaic virus modifies plasmodesmatal size exclusion limit.</a> Science. 246, 377-379 (1989).</li><li>Waigmann, E., Lucas, W. J., Citovsky, V., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+functional+assay+for+tobacco+mosaic+virus+cell-to-cell+movement+protein+and+identification+of+a+domain+involved+in+increasing+plasmodesmal+permeability.">Direct functional assay for tobacco mosaic virus cell-to-cell movement protein and identification of a domain involved in increasing plasmodesmal permeability.</a> Proc. Natl. Acad. Sci. USA. 91, 1433-1437 (1994).</li><li>Ueki, S., Spektor, R., Natale, D. M., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ANK,+a+host+cytoplasmic+receptor+for+the+Tobacco+mosaic+virus+cell-to-cell+movement+protein,+facilitates+intercellular+transport+through+plasmodesmata.">ANK, a host cytoplasmic receptor for the Tobacco mosaic virus cell-to-cell movement protein, facilitates intercellular transport through plasmodesmata.</a> PLoS Pathog. 6, e1001201-e1001201 (2010).</li><li>Coligan, J. E., Dunn, B. M., Ploegh, H. L., Speicher, D. W., Wingfield, P. T., Taylor, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , (2011).</li><li>Tzfira, T., Vaidya, M., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VIP1,+an+Arabidopsis+protein+that+interacts+with+Agrobacterium+VirE2,+is+involved+in+VirE2+nuclear+import+and+Agrobacterium+infectivity.">VIP1, an Arabidopsis protein that interacts with Agrobacterium VirE2, is involved in VirE2 nuclear import and Agrobacterium infectivity.</a> EMBO J. 20, 3596-3607 (2001).</li><li>Chen, M. H., Sheng, J., Hind, G., Handa, A., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+the+tobacco+mosaic+virus+movement+protein+and+host+cell+pectin+methylesterases+is+required+for+viral+cell-to-cell+movement.">Interaction between the tobacco mosaic virus movement protein and host cell pectin methylesterases is required for viral cell-to-cell movement.</a> EMBO J. 19, 913-920 (2000).</li></ol>

इस दृष्टिकोण प्रोटीन के संयोजन के बीच प्रोटीन, प्रोटीन बातचीत, जब कम से कम एक जिनमें से प्रोटीन बंधन बफर में आसानी से घुलनशील है, और 17,18 प्रोटीन के अन्य संयोजन के लिए सफलतापूर्वक लागू किया गया था परीक...

<table border="1"><tbody><tr><td> अभिकर्मक के नाम </td><td> कंपनी </td><td> सूचि संख्या </td></tr><tr><td> प्रोटीन परख किट </td><td> जैव - रेड </td><td> 500-0001 </td></tr><tr><td> Proteinase अवरोध करनेवाला कॉकटेल </td><td> सिग्मा </td><td> S8820 </td></tr><tr><td> मिनी बहुरूपिया सिस्टम </td><td> जैव - रेड </td><td> 165-8000 </td></tr><tr><td> अर्ध - शुष्क पश्चिमी सोख्ता एसडी electrotransfer प्रणाली </td><td> जैव - रेड </td><td> 170-3940 </td></tr><tr><td> विरोधी खरगोश आईजीजी घोड़ा मूली peroxidase के साथ संयुग्मित एंटीबॉडी </td><td> GenScript </td><td> A00098 </td></tr><tr><td> विरोधी जीएसटी खरगोश पॉलीक्लोनल एंटीबॉडी </td><td> GenScript </td><td> A00097 </td></tr><tr><td> एंटी - strepII </td><td> GenScript </td><td> A00626 </td></tr><tr><td> BioTrace, NT nitrocellulose हस्तांतरण झिल्ली </td><td> पाल वैज्ञानिक </td><td> 27377-000 </td></tr><tr><td> Immobilon पश्चिमी chemiluminescent एचआरपी सब्सट्रेट </td><td> Millipore </td><td> S0 050 WBKL </td></tr></tbody></table>

protein membrane overlay assay: a protocol to test interaction between soluble and insoluble proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Watch this Scientific Journal Video about Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro at JoVE.com

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

परीक्षण बातचीत प्रोटीन, प्रोटीन प्रोटीन कार्यक्षमता के विच्छेदन के लिए अपरिहार्य है. यहाँ, हम एक परिचय<em&gt; इन विट्रो में</em&gt; बाध्यकारी प्रोटीन प्रोटीन एक घुलनशील प्रोटीन के साथ एक झिल्ली प्रोटीन immobilized जांच परख. इस परख एक अघुलनशील प्रोटीन और समाधान में एक प्रोटीन के बीच बातचीत का परीक्षण करने के लिए एक विश्वसनीय विधि प्रदान करता है.

प्रोटीन झिल्ली उपरिशायी परख: एक घुलनशील और अघुलनशील प्रोटीन के बीच बातचीत टेस्ट प्रोटोकॉल इन विट्रो में</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

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Biology

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

कार्बनिक सॉल्वेंट और एसिटिक एसिड के बिना जी 250 Coomassie के साथ जैल में प्रोटीन की धुंधला

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

संरचना lipídic Mesophases का उपयोग कर निर्धारण के लिए झिल्ली प्रोटीन crystallizing

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

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

Bimolecular प्याज जीन Helios गन का उपयोग कक्ष में प्रतिदीप्ति complementation (BiFC) प्रोटीन प्रोटीन सहभागिता के लिए परख

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

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

एक तरल चरण आत्मीयता कब्जा चुंबकीय मोती का उपयोग करने के लिए प्रोटीन से प्रोटीन सहभागिता अध्ययन परख: उदाहरण पोलियो वायरस Nanobody

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Lipidic Mesophases में उच्च throughput झिल्ली प्रोटीन का Crystallization के लिए एक रोबोट का प्रयोग

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

झिल्ली macromolecular क्रिस्टलोग्राफी द्वारा संरचना निर्धारण के लिए lipidic Mesophases में बढ़ी प्रोटीन की कटाई और क्रायो ठंडा कण

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

In vitro Methylation Assay to Study Protein Arginine Methylation

In vitro Methylation Assay to Study Protein Arginine Methylation

इन विट्रो मेथिलिकरण परख में प्रोटीन Arginine मेथिलिकरण अध्ययन

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

जीवित कोशिकाओं में प्रोटीन-टुकड़ा complementation परख (पीसीए) द्वारा जीनोम चौड़ा प्रोटीन प्रोटीन सहभागिता स्क्रीनिंग

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

झिल्ली प्रोटीन की हरी फ्लोरोसेंट प्रोटीन आधारित अभिव्यक्ति स्क्रीनिंग में<em&gt; Escherichia कोलाई</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Luciferase निकोटियाना benthamiana में पूरक इमेजिंग परख क्षणिक प्रोटीन प्रोटीन संपर्क गतिशीलता का निर्धारण करने के लिए छोड़ देता है

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...