173 R. Jürgen Dohmen and Martin Scheffner (eds.), Ubiquitin Family Modifiers and the Proteasome: Reviews and Protocols, Methods in Molecular Biology, vol. 832, DOI 10.1007/978-1-61779-474-2_12, © Springer Science+Business Media, LLC 2012 Chapter 12 Isolation of Ubiquitylated Proteins Using Tandem Ubiquitin-Binding Entities Fabienne Aillet, Fernando Lopitz-Otsoa, Roland Hjerpe, Mónica Torres-Ramos, Valérie Lang, and Manuel S. Rodríguez Abstract Studying postubiquitylation events has always been a difficult task due to the labile nature of these post- translational modifications. When utilized in tandem, ubiquitin-binding entities (TUBEs) not only increase up to thousand times the affinity for poly-ubiquitin chains but also protect ubiquitylated proteins from the action of the proteasome and de-ubiquitylating enzymes. Key words: TUBEs, Ubiquitylation, Isolation, Purification, Analysis Purification and enrichment of ubiquitylated proteins is hampered by their inherent instability, stemming both from proteasomal degradation and de-conjugation by de-ubiquitylating enzymes (DUBs) (1, 2). To prevent loss of modified protein, traditional methods rely on the use of tagged ubiquitin, proteasome and DUB- inhibitors, and affinity purification under denaturing conditions (3, 4). While such strategies are powerful methods for pull down of ubiq- uitylated proteins, artifacts related to over expression of tagged ubiquitin or proteasomal inhibition cannot be excluded (5, 6). The exploitation of ubiquitin-binding domains (UBDs) as agarose conjugates for ubiquitin affinity capture circumvents the requirement of tagged ubiquitin (6). However, the generally low affinity of most UBDs for ubiquitin and poly-ubiquitin is limiting and the need for DUB and proteasome inhibitors remains. The tandem ubiquitin-binding domains (TUBEs) were developed to overcome limitations of current tools and methods (7, 8). They consist of tandem UBA domain repeats amino-terminally fused to 1. Introduction