Lb 941 luminometer

Firefly luciferase was analyzed by adding 50 μl Luciferase Assay Reagent (Promega) to 5 μl of translation mixture after cell-free protein synthesis. The concentration of active firefly luciferase was determined using a calibration curve. Luminescence was detected using the LB 941 luminometer (Berthold Technologies). Nanoluciferase (Promega) activity was detected using the Nano-Glo Luciferase Assay System (Promega) according to the manufacturer's instructions. Briefly, 100 μl of reagent was added to 100 μl of cells and incubated for 5 min at 300 rpm. For cell-free produced Nanoluciferase 5 μl translation mixture after cell-free protein synthesis was mixed with 50 μl reagent and incubated for 3 min. Detection of HiBiT (Promega)-tagged eIF2α-S52A in CHO cells was achieved using the Nano-Glo HiBiT Lytic Detection System (Promega) according to the manufacturer's instructions. Briefly, 100 μl Nano-Glo HiBiT Lytic Reagent containing buffer, substrate and LgBiT for luciferase complex formation was added to 100 μl cell suspension and incubation was performed for 10 min at 300 rpm. The luminescence signal of the HiBiT and Nanoluciferase assay was detected by the Multimode Microplate Reader Mithras 2 LB 943 (Berthold Technologies) using an OD2 filter.

Firefly luciferase activity was detected using Luciferase Assay Reagent (Promega, Madison, WI, USA). Therefore, 50 μL of reagent was added to 5 μL of translation mixture after cell-free protein synthesis, and luminescence was detected by the LB 941 luminometer (Berthold Technologies, Bad Wildbad, Germany). The concentration of active luciferase was determined using a calibration curve.