Preparation of the 80S pretermination complex and nanoluciferase release kinetics. (A) Purification of the 80S pretermination nanoluciferase complex stalled on the stop codon in rabbit reticulocyte lysate, using sucrose gradient fractionation. (B) Time-progress curves showing luminescence (in relative luminescence units [RLUs]) of the 80S pretermination nanoluciferase complex upon treatment with recombinant human eRF1 (blue), puromycin (yellow), or buffer (negative control, brown). (C) Time-progress curves showing luminescence (in relative luminescence units [RLUs]) of the 80S pretermination nanoluciferase complex incubated with recombinant yeast eRF1y and treated by yeast eRF3y (green) or buffer (negative control, blue). Exponential fits are shown by black lines. An amount of 20 µL reactions were performed in a microplate reader.
