Leucine incorporation into nascent peptide
This protocol is extracted from research article:
EF-G–induced ribosome sliding along the noncoding mRNA
Sci Adv, Jun 5, 2019; DOI: 10.1126/sciadv.aaw9049

Leucine incorporation experiments were performed by mixing stalled take-off complex (0.1 μM) with ternary complex EF-Tu–GTP–[14C]Leu-tRNALeu (0.3 μM), EF-G (2 μM), and GTP (1 mM) (as indicated). After 2-min incubation at 37°C, samples were quenched with 1/2 volume of 1 M KOH and hydrolyzed for 30 min at 37°C. After neutralization with 1/10 volume of acetic acid, the products were analyzed by high-performance liquid chromatography (HPLC) (LiChrospher 100 RP-8 HPLC column, Merck) using an adapted gradient of acetonitrile in 0.1% trifluoroacetic acid.

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