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Updated: Jan 28, 2026

Detection of Protein Ubiquitination
Published on: August 19, 2009
Detection of ubiquitination activity and identification of ubiquitinated substrates using TR-TUBE
Yukiko Yoshida1, Yasushi Saeki1, Hikaru Tsuchiya1
1Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.
Abstract:
Ubiquitination is a transient posttranslational modification; polyubiquitin chains are removed from proteins by deubiquitinating enzymes (DUBs) and many ubiquitinated proteins are degraded by the proteasome. Exogenously expressed trypsin-resistant tandem ubiquitin-binding entity (TR-TUBE) protects polyubiquitin chains from DUBs and inhibits proteasomal degradation in cells. TR-TUBE effectively binds to substrates ubiquitinated by an exogenously expressed ubiquitin ligase, and enables detection of the specific activity of a given ubiquitin ligase and isolation of its substrates. In this chapter, we describe methods for the detection of ubiquitin ligase activity as well as the identification of substrates of a given ubiquitin ligase using two enrichment tools, TR-TUBE and anti-diGly antibody, coupled with mass spectrometry (MS).
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