A) Unbound Reaction Components. Biotin labeled Ub (bio-Ub) is mixed with GST-tagged E3, E1, E2 and ATP. anti-GST antibody labeled with Eu3+ and streptavidin-APC are then added and excited in the Eu wavelength and the fluorescence of APC is measured. B) Immobilization of Ubiquitin Ligases. GST-tagged E3 is bound to GSH coated magnetic beads. E1, E2, Ub, ATP and compounds are added to the reaction and measured by anti-Ub antibody labeled with the ORIGEN tag that gives a chemiluminescent response. C) Immobilization of Substrate. A high-binding plate is coated with the substrate, unbound substrate is washed off, and the E1, E2, FLAG-Ub, and E3 are added. After unbound proteins are removed anti-FLAG antibody conjugated to HRP is added to the well, washed and HRP substrate is added to detect a signal. D) UBA is immobilized to a high-binding plate. Unbound UBA is removed and E1, E2, E3, Ub, and ATP are added to the well. After the components are removed anti-Ub antibody is added, washed, and secondary antibody conjugated to HRP is added. HRP substrate is added after a final wash step.