moTAP-ANKIB1 was purified from transduced HeLa by using M2 beads followed by streptavidin beads. Once purified, in vitro ubiquitin assay was performed in cell free assay during 1h at 37°C. Purified ANKIB1 was incubated with or without UBE1, UBE2D3, ATP regeneration solution and ubiquitin molecules.