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In vitro translation extracts were made from human 293T cells as described^{23 (link)}. Lysates were nuclease-treated with 18 gel U/μl micrococcal nuclease (NEB M0247S) in the presence of 0.7 mM CaCl₂ for 10 min at 25 °C, and the digestion was stopped by addition of 2.24 mM EGTA. Each translation reaction contained 50% in vitro translation lysate (from 293T cells) and buffer to make the final reaction 0.84 mM ATP, 0.21 mM GTP, 21 mM creatine phosphate (Roche), 45 U/ml creatine phosphokinase (Roche), 10 mM HEPES-KOH, pH 7.6, 2 mM DTT, 8 mM amino acids (Promega), 255 mM spermidine, 1 U/ml murine RNase inhibitor (NEB), and mRNA-specific concentrations of Mg(OAc)2 and KOAc. For 48S ribosome subunit initiation complex purification from in vitro translation reactions, reactions were incubated in the presence of GMP-PNP for 20 min at 30 °C and centrifuged for 6 min at 12 000 × g at 4 °C. Lysates were purified by size-exclusion chromatography through a 1 ml column packed with Sephacryl S-400 gel filtration resin (GE Healthcare) and the eluant centrifuged through a 10–25% (w/v) sucrose gradient by centrifugation for 5 h at 36 000 rpm at 4 °C in a Beckman SW40 Ti rotor. Fractions were collected from the gradient and RNA purified by phenol–chloroform extraction and ethanol precipitation, and protein precipitated with trichloroacetic acid.