UC Davis

The timing and amplitude of plant signaling are frequently regulated through posttranslational modification of key signaling sectors, which facilitates rapid and flexible responses. Protein ubiquitinationUbiquitination can serve as a degradation marker, influence subcellular localization, alter protein-protein interactions, and affect protein activity. Identification of polyubiquitinated proteins has been challenging due to their rapid degradation by the proteasomeProteasome or removal of modifications by deubiquitination enzymes (DUBsDeubiquitinases (DUBs)). Tandem ubiquitin binding entities (TUBEs)Tandem Ubiquitin Binding Entities (TUBEs) are based on ubiquitin-associated domains and protect against both proteasomal degradation and DUBsDeubiquitinases (DUBs). Here, we provide a protocol for purification of ubiquitinated plant proteins using TUBEsTandem Ubiquitin Binding Entities (TUBEs) after transient expressionTransient expression in Nicotiana benthamiana. This protocol can also be applied to other plants to purify multiple ubiquitinated proteins or track ubiquitinationUbiquitination of a target protein. This methodology provides an effective method for identification of ubiquitinUbiquitins ligase substrates and can be coupled with TUBEsTandem Ubiquitin Binding Entities (TUBEs) targeting specific ubiquitinationUbiquitination linkages.