In Vivo Strategies to Isolate and Characterize the Neuronal Ubiquitinated Proteome
Protein ubiquitination is essential for the development of neurons and their proper functioning. Indeed, its failure is associated with a number of neurological disorders. The identification of the proteins that are ubiquitinated in vivo in neurons can greatly contribute to our understanding of the roles that this modification plays in the brain. However, the low stoichiometry at which ubiquitin-modified proteins are found within the cells makes the study of this modification quite challenging.
Here we describe two methodologies that have proven to be suitable approaches for the in vivo analysis of neuronal ubiquitinated proteins. The first approach is based on the in vivo biotinylation of ubiquitin and allows the isolation and enrichment of hundreds of ubiquitin conjugates. The second approach is designed to selectively isolate particular proteins in order to characterize their ubiquitinated fraction.
Key wordsUbiquitination Isolation Neurons Denaturing conditions Biotin-pulldown GFP-pulldown
We would like to acknowledge Maribel Franco and So Young Lee who also contributed to the optimization of these two approaches. We would also like to thank James Sutherland, Rosa Barrio, Michael Clague, Sylvie Urbe, Catherine Lindon, and Jesus Mari Arizmendi for all their advice and support.