An easy tool to monitor the elemental steps of in vitro translation via gel electrophoresis of fluorescently labeled small peptides

Peptides attached to tRNA could be distinguished by urea PAGE following hydrolysis by 1 M NaHCO₃. (A) Intact BPY-M-tRNA^fMet (1) and products of M, MF, and MF2—coding mRNA translation, BPY-M-tRNAfMet (3), BPY-MF-tRNA^Phe (5), BPY-MF₂-tRNA^Phe (7), and the products of their hydrolysis by 1 M NaHCO₃ (2, 4, 6, 8) separated by urea PAGE. (B) Testing the conditions for BPY-M-tRNA^fMet hydrolysis. Lanes correspond to the lack of treatment (1) and treatment with 1 M Na₂CO₃ (2), 1 M NaHCO₃ (3), Tris-HCl (pH 8.0) (4), and Tris (pH 9.0) (5) as indicated. BPY is marked with a green oval—BODIPY label; black circle—methionine; blue oval—tRNA^fMet or tRNA^Phe.