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

Authors
Ramirez, Juanma 
Min, Mingwei 
Barrio, Rosa 
Lindon, Catherine 
Mayor, Ugo 

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Article
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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.

Publication Date
2016
Online Publication Date
2016-09-10
Acceptance Date
2016-06-01
Keywords
Denaturing conditions, Isolation, Substrates, Ubiquitination, Animals, Humans, Proteome, Proteomics, Ubiquitinated Proteins, Ubiquitination
Journal Title
Methods in molecular biology (Clifton, N.J.)
Journal ISSN
1064-3745
1940-6029
Volume Title
1449
Publisher
Springer Nature
Sponsorship
Medical Research Council (MR/M01102X/1)