Luciferase-based real-time translation monitoring assay

Luciferase plasmids were linearized with a blunt-end restriction enzyme just upstream (truncated) or downstream (full-length) of the stop codon, followed by transcription with the mMESSAGE mMACHINE SP6 transcription kit (Invitrogen AM1340). Synthesized mRNA was quantified using a Nanodrop 1000. Nuclease-treated rabbit reticulocyte lysate translation reactions (Promega L4960) were set up in a 384-well plate (Thermo Scientific 164610) on ice. Luciferin (PerkinElmer 122799) was added to each reaction well to a concentration of 0.5 mM followed by 12 units of Superase-In RNase Inhibitor (Invitrogen AM2696). SP6-transcribed truncated or full-length firefly luciferase mRNA was added to a concentration of 40 µg/mL using a multichannel pipette and the plate was immediately inserted into a luminometer microplate reader (Biotek Synergy H1MD) regulated at 30°C. Luminescence readings were taken every few seconds, depending on the number of reaction wells. 5'-guanylyl imidodiphosphate (GDPNP; Jena Bioscience NU-401–50) was added to the wells 16 min after the start of the reaction for 5 min at a concentration of 100 µM followed by a 5-min pretreatment of either 208 µM emetine (Cayman Chemical 21048) or 9.4 µM anisomycin (Sigma A9789). Puromycin (Sigma Aldrich P7255) was added to wells at a concentration of 91 µM. In experiments where GDPNP was not used, the first translation inhibitors were added to the reaction wells at 21 min following the start of the reaction. Reagents were added to the wells by first ejecting the microplate from the luminometer and pipetting the reagents in using a multichannel pipette. The microplate was then promptly inserted again.