In Planta Visualization of Protein Interactions Using Bimolecular Fluorescence Complementation (BiFC).
Identifieur interne : 000676 ( Main/Exploration ); précédent : 000675; suivant : 000677In Planta Visualization of Protein Interactions Using Bimolecular Fluorescence Complementation (BiFC).
Auteurs : Rainer Waadt [Allemagne] ; Jörg KudlaSource :
- CSH protocols ; 2008.
Abstract
INTRODUCTIONBimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein-protein interactions in living cells. This method has been successfully adapted to a variety of expression systems in different organisms. BiFC is based on the formation of a fluorescent complex by fragments of the enhanced yellow fluorescent protein (eYFP) when brought together by the interaction of two associating proteins fused to these fragments. Interaction of these proteins restores fluorescence and allows the visualization of spatial localization patterns of protein complexes. Absence of interaction prevents reassembly of the fluorescent protein and results only in background fluorescence. The specificity of bimolecular fluorescence complementation must be confirmed by parallel analysis of proteins in which the interaction interface has been mutated. This protocol describes the Agrobacterium-mediated transient expression protocol for BiFC assays in Nicotiana benthamiana leaf cells. This method exhibits a high transformation rate (up to 90% of the cells) and allows the simultaneous expression of multiple proteins in single cells. Therefore, this expression system enables colocalization analyses of fluorescently labeled proteins with the formation of BiFC complexes for determination of cellular complex localization. In addition, protein interaction assays in N. benthamiana leaves permit the investigation of protein interactions at different time points of expression, allow analysis of proteins that are normally toxic in protoplasts, and enable comparative protein interaction investigation in epidermal cells as well as in mesophyll protoplasts.
DOI: 10.1101/pdb.prot4995
PubMed: 21356813
Affiliations:
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This method has been successfully adapted to a variety of expression systems in different organisms. BiFC is based on the formation of a fluorescent complex by fragments of the enhanced yellow fluorescent protein (eYFP) when brought together by the interaction of two associating proteins fused to these fragments. Interaction of these proteins restores fluorescence and allows the visualization of spatial localization patterns of protein complexes. Absence of interaction prevents reassembly of the fluorescent protein and results only in background fluorescence. The specificity of bimolecular fluorescence complementation must be confirmed by parallel analysis of proteins in which the interaction interface has been mutated. This protocol describes the Agrobacterium-mediated transient expression protocol for BiFC assays in Nicotiana benthamiana leaf cells. This method exhibits a high transformation rate (up to 90% of the cells) and allows the simultaneous expression of multiple proteins in single cells. Therefore, this expression system enables colocalization analyses of fluorescently labeled proteins with the formation of BiFC complexes for determination of cellular complex localization. In addition, protein interaction assays in N. benthamiana leaves permit the investigation of protein interactions at different time points of expression, allow analysis of proteins that are normally toxic in protoplasts, and enable comparative protein interaction investigation in epidermal cells as well as in mesophyll protoplasts.</div></front></TEI><pubmed><MedlineCitation Status="PubMed-not-MEDLINE" Owner="NLM"><PMID Version="1">21356813</PMID><DateCompleted><Year>2011</Year><Month>07</Month><Day>14</Day></DateCompleted><DateRevised><Year>2020</Year><Month>09</Month><Day>29</Day></DateRevised><Article PubModel="Electronic"><Journal><JournalIssue CitedMedium="Internet"><Volume>2008</Volume><PubDate><Year>2008</Year><Month>Apr</Month><Day>01</Day></PubDate></JournalIssue><Title>CSH protocols</Title><ISOAbbreviation>CSH Protoc</ISOAbbreviation></Journal><ArticleTitle>In Planta Visualization of Protein Interactions Using Bimolecular Fluorescence Complementation (BiFC).</ArticleTitle><Pagination><MedlinePgn>pdb.prot4995</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1101/pdb.prot4995</ELocationID><ELocationID EIdType="pii" ValidYN="Y">2008/4/pdb.prot4995</ELocationID><Abstract><AbstractText>INTRODUCTIONBimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein-protein interactions in living cells. This method has been successfully adapted to a variety of expression systems in different organisms. BiFC is based on the formation of a fluorescent complex by fragments of the enhanced yellow fluorescent protein (eYFP) when brought together by the interaction of two associating proteins fused to these fragments. Interaction of these proteins restores fluorescence and allows the visualization of spatial localization patterns of protein complexes. Absence of interaction prevents reassembly of the fluorescent protein and results only in background fluorescence. The specificity of bimolecular fluorescence complementation must be confirmed by parallel analysis of proteins in which the interaction interface has been mutated. This protocol describes the Agrobacterium-mediated transient expression protocol for BiFC assays in Nicotiana benthamiana leaf cells. This method exhibits a high transformation rate (up to 90% of the cells) and allows the simultaneous expression of multiple proteins in single cells. Therefore, this expression system enables colocalization analyses of fluorescently labeled proteins with the formation of BiFC complexes for determination of cellular complex localization. In addition, protein interaction assays in N. benthamiana leaves permit the investigation of protein interactions at different time points of expression, allow analysis of proteins that are normally toxic in protoplasts, and enable comparative protein interaction investigation in epidermal cells as well as in mesophyll protoplasts.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Waadt</LastName><ForeName>Rainer</ForeName><Initials>R</Initials><AffiliationInfo><Affiliation>Institut für Botanik und Botanischer Garten, Universität Münster, 48149 Münster, Germany.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Kudla</LastName><ForeName>Jörg</ForeName><Initials>J</Initials></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2008</Year><Month>04</Month><Day>01</Day></ArticleDate></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>CSH Protoc</MedlineTA><NlmUniqueID>101280522</NlmUniqueID><ISSNLinking>1559-6095</ISSNLinking></MedlineJournalInfo></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="entrez"><Year>2011</Year><Month>3</Month><Day>2</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2008</Year><Month>1</Month><Day>1</Day><Hour>0</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2008</Year><Month>1</Month><Day>1</Day><Hour>0</Hour><Minute>1</Minute></PubMedPubDate></History><PublicationStatus>epublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">21356813</ArticleId><ArticleId IdType="doi">10.1101/pdb.prot4995</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>Allemagne</li></country><region><li>District de Münster</li><li>Rhénanie-du-Nord-Westphalie</li></region><settlement><li>Münster</li></settlement></list><tree><noCountry><name sortKey="Kudla, Jorg" sort="Kudla, Jorg" uniqKey="Kudla J" first="Jörg" last="Kudla">Jörg Kudla</name></noCountry><country name="Allemagne"><region name="Rhénanie-du-Nord-Westphalie"><name sortKey="Waadt, Rainer" sort="Waadt, Rainer" uniqKey="Waadt R" first="Rainer" last="Waadt">Rainer Waadt</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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