Protein Ubiquitination Assay

Ubiquitin, a 76-amino acid protein which could covalently bind to a target protein, is catalyzed by an enzymatic cascade comprising of ATP-dependent ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin-protein ligase E3. Histidine (His) pull-down and immunoprecipitation (IP) assays are frequently adopted to detect FOXP3 ubiquitination, which is one of the most important posttranslational modifications and may induce signal transduction or 26S proteasome-dependent degradation of FOXP3 in various immune milieus.