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Isolation of Ubiquitinated Proteins to High Purity from In Vivo Samples.

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cam.issuedOnline2016-09-10
dc.contributor.authorRamirez, Juanma
dc.contributor.authorMin, Mingwei
dc.contributor.authorBarrio, Rosa
dc.contributor.authorLindon, Catherine
dc.contributor.authorMayor, Ugo
dc.contributor.orcidLindon, Cath [0000-0003-3554-2574]
dc.date.accessioned2018-10-31T06:51:18Z
dc.date.available2018-10-31T06:51:18Z
dc.date.issued2016
dc.description.abstractUbiquitination pathways are widely used within eukaryotic cells. The complexity of ubiquitin signaling gives rise to a number of problems in the study of specific pathways. One problem is that not all processes regulated by ubiquitin are shared among the different cells of an organism (e.g., neurotransmitter release is only carried out in neuronal cells). Moreover, these processes are often highly temporally dynamic. It is essential therefore to use the right system for each biological question, so that we can characterize pathways specifically in the tissue or cells of interest. However, low stoichiometry, and the unstable nature of many ubiquitin conjugates, presents a technical barrier to studying this modification in vivo. Here, we describe two approaches to isolate ubiquitinated proteins to high purity. The first one favors isolation of the whole mixture of ubiquitinated material from a given tissue or cell type, generating a survey of the ubiquitome landscape for a specific condition. The second one favors the isolation of just one specific protein, in order to facilitate the characterization of its ubiquitinated fraction. In both cases, highly stringent denaturing buffers are used to minimize the presence of contaminating material in the sample.
dc.identifier.doi10.17863/CAM.31810
dc.identifier.eissn1940-6029
dc.identifier.issn1064-3745
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/284438
dc.languageeng
dc.language.isoeng
dc.publisherSpringer Nature
dc.publisher.urlhttps://link.springer.com/protocol/10.1007/978-1-4939-3756-1_10#Abs1
dc.subjectDenaturing conditions
dc.subjectIsolation
dc.subjectSubstrates
dc.subjectUbiquitination
dc.subjectAnimals
dc.subjectHumans
dc.subjectProteome
dc.subjectProteomics
dc.subjectUbiquitinated Proteins
dc.subjectUbiquitination
dc.titleIsolation of Ubiquitinated Proteins to High Purity from In Vivo Samples.
dc.typeArticle
dcterms.dateAccepted2016-06-01
prism.endingPage202
prism.publicationNameMethods in molecular biology (Clifton, N.J.)
prism.startingPage193
prism.volume1449
pubs.funder-project-idMedical Research Council (MR/M01102X/1)
rioxxterms.licenseref.startdate2016-06-01
rioxxterms.licenseref.urihttp://www.rioxx.net/licenses/all-rights-reserved
rioxxterms.typeJournal Article/Review
rioxxterms.versionAM
rioxxterms.versionofrecord10.1007/978-1-4939-3756-1_10

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