Figure 1.

Translation in human cell-free extract. (A) Schematic representation of the individual steps from human HeLa cell culture to a cell-free human in vitro translation extract. Cells were grown in a large spinner flask. After harvesting and washing of the cells, they were subjected to nitrogen pressure and disrupted by sudden pressure release. A quick centrifugation step resulted in a crude extract which can be supplemented for optimal performance of mRNA in vitro translation. Example of in vitro translational stalling optimization for (B) time (C) magnesium acetate concentration and (D) potassium acetate concentration using Western blotting with anti-HA antibody for detection of tRNA-bound hCMV peptide or free hCMV peptide. Optimal conditions vary for each extract.