Identification of Connexin-Interacting Proteins: Application of the Yeast Two-Hybrid Screen
Protein–protein interactions are recognized as one of the fundamental mechanisms for relaying the intra- and intercellular signals that are required for normal cellular activities affecting growth, development, and maintenance of homeostasis in tissues and organs. The yeast two-hybrid screen has bec...
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| Veröffentlicht in: | Methods (San Diego, Calif.) Calif.), 2000-02, Vol.20 (2), p.219-231 |
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| creator | Jin, Chengshi Lau, Alan F. Martyn, Kendra Dean |
| description | Protein–protein interactions are recognized as one of the fundamental mechanisms for relaying the intra- and intercellular signals that are required for normal cellular activities affecting growth, development, and maintenance of homeostasis in tissues and organs. The yeast two-hybrid screen has become a valuable tool for identifying protein–protein interactions. The gap junction protein connexin 43 (Cx43) has been implicated in a number of biological processes including development and cellular growth control. To further advance our understanding of the ways in which Cx43 may influence these cellular activities, and to extend our knowledge of the regulation of Cx43 function and/or processing, we have employed the yeast two-hybrid screen technique to identify Cx43-interacting proteins. We present detailed methods for the yeast two-hybrid screen of a mouse embryonic cDNA library using the C terminus of Cx43 as “bait.” We also describe additional methods to confirm the interactions between Cx43 and the identified proteins. These methods include in vitro binding assays, coimmunoprecipitation, and subcellular localization using immunofluorescence microscopy. |
| doi_str_mv | 10.1006/meth.1999.0939 |
| format | Article |
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The yeast two-hybrid screen has become a valuable tool for identifying protein–protein interactions. The gap junction protein connexin 43 (Cx43) has been implicated in a number of biological processes including development and cellular growth control. To further advance our understanding of the ways in which Cx43 may influence these cellular activities, and to extend our knowledge of the regulation of Cx43 function and/or processing, we have employed the yeast two-hybrid screen technique to identify Cx43-interacting proteins. We present detailed methods for the yeast two-hybrid screen of a mouse embryonic cDNA library using the C terminus of Cx43 as “bait.” We also describe additional methods to confirm the interactions between Cx43 and the identified proteins. 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Lau, Alan F. ; Martyn, Kendra Dean</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c340t-9c8ddbff0ceb85fef0d3017b8caa2b08e875ad9f845d2b283f1509d2faf7a7e53</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2000</creationdate><topic>Animals</topic><topic>Bacterial Proteins - genetics</topic><topic>Bacterial Proteins - metabolism</topic><topic>beta-Galactosidase - genetics</topic><topic>beta-Galactosidase - metabolism</topic><topic>Connexin 43 - genetics</topic><topic>Connexin 43 - metabolism</topic><topic>DNA-Binding Proteins</topic><topic>Embryo, Mammalian</topic><topic>Escherichia coli - genetics</topic><topic>Fungal Proteins - genetics</topic><topic>Fungal Proteins - metabolism</topic><topic>Gap Junctions - physiology</topic><topic>Gene Library</topic><topic>Gene Transfer Techniques</topic><topic>Mice</topic><topic>Plasmids</topic><topic>Promoter Regions, Genetic</topic><topic>Recombinant Fusion Proteins - metabolism</topic><topic>Repressor Proteins</topic><topic>Restriction Mapping</topic><topic>Saccharomyces cerevisiae - genetics</topic><topic>Saccharomyces cerevisiae Proteins</topic><topic>Serine Endopeptidases - genetics</topic><topic>Serine Endopeptidases - metabolism</topic><topic>Transcription Factors - genetics</topic><topic>Transcription Factors - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Jin, Chengshi</creatorcontrib><creatorcontrib>Lau, Alan F.</creatorcontrib><creatorcontrib>Martyn, Kendra Dean</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><jtitle>Methods (San Diego, Calif.)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Jin, Chengshi</au><au>Lau, Alan F.</au><au>Martyn, Kendra Dean</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Identification of Connexin-Interacting Proteins: Application of the Yeast Two-Hybrid Screen</atitle><jtitle>Methods (San Diego, Calif.)</jtitle><addtitle>Methods</addtitle><date>2000-02-01</date><risdate>2000</risdate><volume>20</volume><issue>2</issue><spage>219</spage><epage>231</epage><pages>219-231</pages><issn>1046-2023</issn><eissn>1095-9130</eissn><abstract>Protein–protein interactions are recognized as one of the fundamental mechanisms for relaying the intra- and intercellular signals that are required for normal cellular activities affecting growth, development, and maintenance of homeostasis in tissues and organs. The yeast two-hybrid screen has become a valuable tool for identifying protein–protein interactions. The gap junction protein connexin 43 (Cx43) has been implicated in a number of biological processes including development and cellular growth control. To further advance our understanding of the ways in which Cx43 may influence these cellular activities, and to extend our knowledge of the regulation of Cx43 function and/or processing, we have employed the yeast two-hybrid screen technique to identify Cx43-interacting proteins. We present detailed methods for the yeast two-hybrid screen of a mouse embryonic cDNA library using the C terminus of Cx43 as “bait.” We also describe additional methods to confirm the interactions between Cx43 and the identified proteins. 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| fulltext | fulltext |
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| ispartof | Methods (San Diego, Calif.), 2000-02, Vol.20 (2), p.219-231 |
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| source | MEDLINE; ScienceDirect Journals (5 years ago - present) |
| subjects | Animals Bacterial Proteins - genetics Bacterial Proteins - metabolism beta-Galactosidase - genetics beta-Galactosidase - metabolism Connexin 43 - genetics Connexin 43 - metabolism DNA-Binding Proteins Embryo, Mammalian Escherichia coli - genetics Fungal Proteins - genetics Fungal Proteins - metabolism Gap Junctions - physiology Gene Library Gene Transfer Techniques Mice Plasmids Promoter Regions, Genetic Recombinant Fusion Proteins - metabolism Repressor Proteins Restriction Mapping Saccharomyces cerevisiae - genetics Saccharomyces cerevisiae Proteins Serine Endopeptidases - genetics Serine Endopeptidases - metabolism Transcription Factors - genetics Transcription Factors - metabolism |
| title | Identification of Connexin-Interacting Proteins: Application of the Yeast Two-Hybrid Screen |
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