EMSA of tRNA Binding to TrhO

EMSA was performed essentially as described^{34 (link)}. Recombinant TrhO (0, 10, 20, 40, or 80 pmol) and in vitro transcribed tRNA^Ala1 or tRNA^Leu3 (20 pmol each) were incubated at 37 °C for 1 h in 10 µl of reaction mixture [50 mM HEPES-KOH (pH 7.5), 5 mM Mg(OAc)₂, 100 mM KCl, 1 mM spermine, 1 mM DTT]. The mixtures were electrophoresed in 6% native polyacrylamide gel with running buffer [50 mM HEPES-KOH (pH 7.5), 5 mM Mg(OAc)₂, and 1 mM DTT] in cold room. The gel was first stained with SYBR Safe (Thermo Fisher Scientific) to detect tRNA and then with Coomassie brilliant blue (Nacalai Tesque) to detect protein.