Isolation of Ubiquitinated Proteins to High Purity from In Vivo Samples
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.
Key wordsUbiquitination Substrates Isolation Denaturing conditions
While this chapter was written by the authors listed above, we would like to acknowledge other lab members who contributed to optimization of these techniques as described: Our thanks therefore to Maribel Franco, James Sutherland, Aitor Martinez, Benoit Lectez, and So Young Lee. We would also like to thank Junmin Peng and Gunnar Dittmar, without whose excellent MS support we would never have confirmed how well our bioUb pulldown strategy was performing. The authors would like to acknowledge networking support by the Proteostasis COST Action (BM1307).
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