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Ubiquitin can be added to substrate protein as a protein tag by the concerted actions of ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin protein ligase (E3). At the present of E1 and ubiquitin, E2 activity can be determined by the thio-ester formation. The E3 activity of a putative protein as well as the E2/E3 or E3/substrate specificities also can be explored by in vitro ubiquitination assay. The result can be detected by western blot with certain antibody. Purified proteins expressed from bacterial system are always used in this assay.