Isolation of Ubiquitinated Proteins to High Purity from In Vivo Samples.
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Publication Date
2016Journal Title
Methods in molecular biology (Clifton, N.J.)
ISSN
1064-3745
Publisher
Springer Nature
Volume
1449
Pages
193-202
Language
eng
Type
Article
Metadata
Show full item recordCitation
Ramirez, J., Min, M., Barrio, R., Lindon, C., & Mayor, U. (2016). Isolation of Ubiquitinated Proteins to High Purity from In Vivo Samples.. Methods in molecular biology (Clifton, N.J.), 1449 193-202. https://doi.org/10.1007/978-1-4939-3756-1_10
Abstract
Ubiquitination 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.
Keywords
Denaturing conditions, Isolation, Substrates, Ubiquitination, Animals, Humans, Proteome, Proteomics, Ubiquitinated Proteins, Ubiquitination
Sponsorship
Medical Research Council (MR/M01102X/1)
Identifiers
External DOI: https://doi.org/10.1007/978-1-4939-3756-1_10
This record's URL: https://www.repository.cam.ac.uk/handle/1810/284438
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