DC Field | Value | Language |
---|---|---|
dc.contributor.author | Lee, Hong-Won | ko |
dc.contributor.author | Ryu, Ji Young | ko |
dc.contributor.author | Yoo, Janghyun | ko |
dc.contributor.author | Choi, Byungsan | ko |
dc.contributor.author | Kim, Kipom | ko |
dc.contributor.author | Yoon, Tae-Young | ko |
dc.date.accessioned | 2014-08-28T08:27:27Z | - |
dc.date.available | 2014-08-28T08:27:27Z | - |
dc.date.created | 2013-11-25 | - |
dc.date.created | 2013-11-25 | - |
dc.date.issued | 2013-10 | - |
dc.identifier.citation | NATURE PROTOCOLS, v.8, no.10, pp.2045 - 2060 | - |
dc.identifier.issn | 1754-2189 | - |
dc.identifier.uri | http://hdl.handle.net/10203/188532 | - |
dc.description.abstract | Coimmunoprecipitation (co-IP) analysis is a useful method for studying protein-protein interactions. It currently involves electrophoresis and western blotting, which are not optimized for detecting weak and transient interactions. In this protocol we describe an advanced version of co-IP analysis that uses real-time, single-molecule fluorescence imaging as its detection scheme. Bait proteins are pulled down onto the imaging plane of a total internal reflection (TIR) microscope. With unpurified cells or tissue extracts kept in reaction chambers, we observe single protein-protein interactions between the surface-immobilized bait and the fluorescent protein-labeled prey proteins in real time. Such direct recording provides an improvement of five orders of magnitude in the time resolution of co-IP analysis. With the single-molecule sensitivity and millisecond time resolution, which distinguish our method from other methods for measuring weak protein-protein interactions, it is possible to quantify the interaction kinetics and active fraction of native, unlabeled bait proteins. Real-time single-molecule co-IP analysis, which takes similar to 4 h to complete from lysate preparation to kinetic analysis, provides a general avenue for revealing the rich kinetic picture of target protein-protein interactions, and it can be used, for example, to investigate the molecular lesions that drive individual cancers at the level of protein-protein interactions. | - |
dc.language | English | - |
dc.publisher | NATURE PUBLISHING GROUP | - |
dc.subject | PROXIMITY LIGATION | - |
dc.subject | PULL-DOWN | - |
dc.subject | IN-SITU | - |
dc.subject | RAS | - |
dc.subject | BINDING | - |
dc.subject | RECEPTOR | - |
dc.subject | DYNAMICS | - |
dc.subject | KINASES | - |
dc.subject | CANCER | - |
dc.subject | DOMAIN | - |
dc.title | Real-time single-molecule coimmunoprecipitation of weak protein-protein interactions | - |
dc.type | Article | - |
dc.identifier.wosid | 000326163400015 | - |
dc.identifier.scopusid | 2-s2.0-84885013241 | - |
dc.type.rims | ART | - |
dc.citation.volume | 8 | - |
dc.citation.issue | 10 | - |
dc.citation.beginningpage | 2045 | - |
dc.citation.endingpage | 2060 | - |
dc.citation.publicationname | NATURE PROTOCOLS | - |
dc.identifier.doi | 10.1038/nprot.2013.116 | - |
dc.embargo.liftdate | 9999-12-31 | - |
dc.embargo.terms | 9999-12-31 | - |
dc.contributor.localauthor | Yoon, Tae-Young | - |
dc.contributor.nonIdAuthor | Lee, Hong-Won | - |
dc.contributor.nonIdAuthor | Ryu, Ji Young | - |
dc.contributor.nonIdAuthor | Yoo, Janghyun | - |
dc.contributor.nonIdAuthor | Choi, Byungsan | - |
dc.type.journalArticle | Article | - |
dc.subject.keywordPlus | PROXIMITY LIGATION | - |
dc.subject.keywordPlus | PULL-DOWN | - |
dc.subject.keywordPlus | IN-SITU | - |
dc.subject.keywordPlus | RAS | - |
dc.subject.keywordPlus | BINDING | - |
dc.subject.keywordPlus | RECEPTOR | - |
dc.subject.keywordPlus | DYNAMICS | - |
dc.subject.keywordPlus | KINASES | - |
dc.subject.keywordPlus | CANCER | - |
dc.subject.keywordPlus | DOMAIN | - |
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