Western Blot of WT <t>GFP</t> and GFP Y39TAG mutant expression in a cell-free translation system. Synthesis of WT GFP and the GFP Y39TAG mutant was performed using the <t>RTS</t> E. coli HY Kit, to which the corresponding plasmid (500 µg/mL), purified Mj TyrRS and cognate suppressor Mj tRNA CUA (tRNA) or T-stem modified tRNA CUA Opt (denoted as *) were added. (A) Expression of WT GFP and the GFP Y39TAG mutant in the presence of Mj TyrRS (300 µg/mL) and synthetic Mj tRNA CUA (60 µg/mL). The band at 28 kDa corresponds to full-length GFP. (B) Western blot analysis demonstrates enhanced GFP Y39TAG protein expression as a function of increased Mj TyrRS concentrations in a cell-free reaction medium supplied with Mj tRNA CUA (60 µg/mL – top panel and 450 µg/mL – bottom panel). ( C ) Dependence of GFP Y39TAG yield on the type and concentration of nonsense suppressor, as visualized by Western blot.