Figure 4.

Real-time kinetics of FRET IκBα diubiquitination under single-round turnover condition. The reaction was carried out as in Figure 3, A–C expect that the preformed substrate–E3 and donor Ub–E2 complexes were treated with apyrase (6 mU/1 μl) for 1 min at 37 °C to deplete ATP. The apyrase-treated reaction mixture was then loaded onto a 384-well plate for incubation on the Synergy-H1 reader set at 30 °C. The reaction was monitored at times as indicated. The emission spectrum of 544 to 700 nm for the complete reaction and no E1 control are shown. Ub, ubiquitin.