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. 2019 Apr 16;2(2):30. doi: 10.3390/mps2020030

Figure 2.

Figure 2

Optimization strategies for eukaryotic cell-free systems. (A) Schematic overview of strategies for improvement of eukaryotic cell-free systems concerning protein quality and quantity. (B) PathScan® Phospho-eIF2α (Ser51) Sandwich ELISA (Cell Signaling) for the detection of eIF2α-P in CHO cell-free reactions. Untreated control (UTC), DMSO treated samples (DMSO) and C38 treated samples were analyzed for the presence of eIF2α-P. ELISA was performed according to the manufacturer´s protocol. (C) Western Blot analysis of UTC, DMSO treated samples (DMSO) and C38 treated samples using a primary anti-eIF2α-P antibody (Cell Signaling) and a secondary anti-rabbit-HRP antibody. Western blot signal was detected using ECL reagent (Promega) and a Typhoon Trio Plus Imager (GE Healthcare). Image analysis was performed using ImageQuant TL software (GE Healthcare). (D) Eukaryotic cell-free reaction in the presence of C38 PERK inhibitor. CHO lysate based cell-free reaction was performed using pIX4.0-Luc plasmid (Luciferase) in the presence of various concentrations (1 mM up to 6 mM) of small component C38. Production of cell-free synthesized Luciferase was analyzed using a standard Luciferase assay (Promega).