Fig. 5

Real-time tracking of a ubiquitination cascade in a single well. a Schematic of the multi-step ubiquitination assay. Ube1 immobilized on small beads and E6AP immobilized on large beads were placed together in one well with a fluorescent Ub. In the presence of ATP, ubiquitin was loaded onto Ube1 (1), forming a fluorescent ring on small beads. Upon addition of Ube2L3 to the solution, ubiquitin was transferred to Ube2L3 and a decrease in ring intensity on small beads (Ube1) was observed (2). Simultaneously, as Ube2L3 further transfers the fluorescent Ub to E6AP, ring intensity on large beads increased (3). b The reaction was monitored by imaging on the confocal scanning microscope Opera™ (Perkin Elmer) over time. Images were acquired in the brightfield (LED) and Cy5 fluorescence emission detection channels (640 nm). Ring intensities from small (Ube1) and large (E6AP) beads were analyzed as described in “Methods”. Ubiquitin loading on Ube1 (blue line) and conjugation on E6AP (red line) which occurred simultaneously in each well are represented in charts, corresponding to wells as indicated: b with ATP and Ube2L3 (+ ATP + Ube2L3), c without Ube2L3 (+ATP − Ube2L3), and d without ATP (−ATP + Ube2L3). Shown are exemplary images at the start of experiment (0), after adding ATP (1), and after 2-h incubation (3). Over time, the intensity of the Cy5-Ub conjugates decreased on Ube1 beads and increased on the E6AP beads