Quantifying Cell-Free Protein Yields

Determination of total protein yields was performed in triplicates based on liquid scintillation as described previously (Stech et al., 2017 (link)). Following cell-free protein synthesis and fractionation into SN1 and SN2, aliquots were withdrawn from the solution, mixed with 3 ml trichloroacetic acid (TCA), and incubated in an 80°C water bath for 15 min, followed by incubation on ice for 30 min. In order to remove non-incorporated ¹⁴C-leucine, protein solutions were filtered using a vacuum filtration system (Hoefer, Holliston, MA, United States) and glass fiber filters (Machery-Nagel, Düren, Germany). Incorporation of ¹⁴C-leucine in cell-free synthesized proteins was measured by liquid scintillation counting using the HIDEX 600 SL (Hidex, Turku, Finland).