In E. coli, the pACYC-HA-Ub-E1 (UBA1)-UbcH5A-RNF126 and pET-22b-MIDN-His₆ plasmids were co-transformed. After IPTG induction to express all proteins required for ubiquitination, His-tagged MIDN was enriched with Ni-NTA resin and subjected to mass-spectrometric mapping of its ubiquitin-modified sites.