Typhoon fla 9500 phosphorimager system

Reactions containing 2 nM initiation complexes and 1 μM eIF5A in 1X Buffer E (20 mM Tris pH 7.5, 100 mM KOAc pH 7.6, 2.5 mM Mg(OAc)₂, 0.25 mM Spermidine, and 2 mM DTT) were incubated at 26°C in the presence of 5 mM puromycin. Time points over the course of 90 min were quenched with 250 mM KOH and analyzed by electrophoretic TLC (Millipore). TLC plates were equilibrated with pyridine acetate buffer (5 mL pyridine, 200 mL acetic acid in 1 l, pH 2.8) before electrophoresis at 1200 V for 15min. Plates were developed using a Typhoon FLA 9500 Phosphorimager system (GE Healthcare Life Sciences) and quantified using ImageQuantTL (GE Healthcare Life Sciences). Time courses were fit to single exponential kinetics using Kaleidagraph (Synergy Software).

Translation elongation reactions were performed in 1X Buffer E (20 mM Tris pH 7.5, 100 mM KOAc pH 7.6, 2.5 mM Mg(OAc)₂, 0.25 mM Spermidine, and 2 mM DTT). Limited amounts of 80S initiation complexes (3 nM) were mixed with purified eEF1A (1 μM), aa-tRNA (500 nM), eEF2 (1 μM), eEF3 (1 μM), ATP (3 mM) and GTP (2 mM) in the presence or absence of eIF5A (1 μM). Reactions were incubated at 26°C and time points quenched with 250 mM KOH. Peptide formation was monitored by electrophoretic TLC (Millipore). TLC plates were equilibrated with pyridine acetate buffer (5 mL pyridine, 200 mL acetic acid in 1 l, pH 2.8) before electrophoresis at 1200 V for 28 min. Plates were developed using a Typhoon FLA 9500 Phosphorimager system (GE Healthcare Life Sciences) and quantified using ImageQuantTL (GE Healthcare Life Sciences). Time courses were fit to single exponential kinetics using Kaleidagraph (Synergy Software).