Fig. 3.

Ubiquitination activity of E1, E2, and HECT E3 enzymes detected over time using UPS-CONA. His6-tagged enzymes Ube1 (a), Ube2L3 (b), or E6AP (c) were immobilized on Ni²⁺NTA agarose microbeads, placed in a 384-well plate and incubated in the reaction buffer at 37 °C in the presence of Cy5-labeled ubiquitin (a), Cy5-labeled ubiquitin and untagged Ube1 (b) or Cy5-labeled ubiquitin, untagged Ube1 and Ube2L3 (c). Reactions without ATP (− ATP) were used as controls for non-enzymatic binding of Cy5-Ub to the beads. Reactions were performed in triplicates. At the end of each reaction, DTT was added to remove thioester-bound Cy5-Ub from the on-bead enzymes to quantify non-thioester-bound fraction. Images were acquired on the confocal scanning microscope Opera™ (Perkin Elmer), in brightfield for detection of beads and in Cy5 fluorescence emission for detection of Cy5-Ub conjugates. Data were analyzed as described in “Methods”. Average ring intensities in each well over time are represented, corresponding to Cy5-Ub conjugated to the on-bead enzyme