5.3.5. Assays of Cell-Free Protein Synthesis

The effect of RIPs on protein synthesis was determined through a coupled transcription–translation in vitro assay using a rabbit reticulocyte lysate system [13]. The reaction mixture contained 0.6 μL of rabbit reticulocyte lysate and 5.8 μL of a mixture of the following components: 4.6 U ribonuclease inhibitor, 2.3 U T7 RNA polymerase, 0.2 μg luciferase T7 plasmid, rNTPs (0.4 mM each), amino acids (2 μM each), 10 mM Tris–HCl (pH 7.8), 0.2 mM spermidine, 28 mM KCl, 1 mM MgCl₂, and nuclease-free water. The mixtures were incubated at 30 °C for 10 min and placed on ice. Then, 1.6 μL of either water or different protein concentrations were added and the sample mixture was incubated at 30 °C for 40 min. Subsequently, 25 μL water was added and mixed with 28 μL of Luciferase Assay Reagent (Promega, Alcobendas, Madrid, Spain) at room temperature. Luminescence was determined with a Junior LB 9509 luminometer (Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany). Three experiments were conducted in duplicate, and IC₅₀ (concentration that inhibits 50% protein synthesis) values were calculated by linear regression.