Assembly of different length of polyubiquitins on the catalytic cysteine of E2 enzymes without E3 ligase; a novel application of non-reduced/reduced 2-dimensional electrophoresis

► NR/R-2DE as a powerful tool to analyze thioester-linked ubiquitins. ► E3-independent ubiquitination by Ube2K, Cdc34, Ube2g2, and UbcH10. ► Assembly of different length of ubiquitin chains on catalytic cysteine of E2s. ► Preassembly of polyubiquitin on E2 supports the possibility of 'en bloc transfer'.