Retic lysate ivt kit

In vitro translation assays were performed with the Retic Lysate IVT Kit (Invitrogen-AM1200, Villebon Sur Yvette, France) following the manufacturer’s instructions and using the F-Luc mRNA (12.5 ng/µL) as a reporter. The translation efficiency of this transcript was analyzed in the presence of either P. lividus eIF4B or R-Luc mRNAs, both at a final concentration of 12.5 ng/µL. Incubations were performed at 30 °C in an Eppendorf ThermoMixer with agitation at 300 rpm. For each time point, 10 µL of the reaction assay was mixed with 50 µL of ONE-Glo reagent (Promega-E6110, Charbonnières-les-Bains, France), and the sample luminescence was measured for 10 s on a 96-well microplate using a Tristar luminometer (Berthold).

For in vitro translation, the Retic Lysate IVT kit (Invitrogen), a eukaryotic cell-free protein expression system, was used. In a total volume of 15 µl, 40 ng of the FLuc–mRNA (capped as indicated), 50 µM l-methionine and 150 mM potassium acetate were mixed with 8.5 µl of the reticulocyte lysate and incubated for 90 min at 30 °C. Samples were mixed with 8.5 µl of the reticulocyte lysate and incubated for 90 min at 30 °C. Afterwards, 2 µl of the respective translation mix was further used in a luminescence assay. The translation efficiencies of the differently capped FLuc–mRNAs were measured using a luciferase assay based on the Beetle-juice Luciferase Assay Firefly (pjk). Luciferase activity was determined after adding 50 µl of freshly prepared substrate solution to the translation mixture. Luminescence was assessed using a Tecan Infinite M1000 PRO microplate reader with an integration time of 3 s. Differently capped mRNAs were used. ApppG-capped mRNA represents cap-independent translation and was subtracted as background from the other samples. All values were normalized to m⁷GpppG-capped mRNA.