Author: Jaïs, Philippe H; Decroly, Etienne; Jacquet, Eric; Le Boulch, Marine; Jaïs, Aurélien; Jean-Jean, Olivier; Eaton, Heather; Ponien, Prishila; Verdier, Fréderique; Canard, Bruno; Goncalves, Sergio; Chiron, Stéphane; Le Gall, Maude; Mayeux, Patrick; Shmulevitz, Maya
Title: C3P3-G1: first generation of a eukaryotic artificial cytoplasmic expression system Document date: 2019_3_18
ID: 6nq7y1qe_22
Snippet: GTase assay. The pppAC 4 RNA was synthetized using T7 DNA primase as described by Peyrane et al. (8) . The RNA (pppAC 4 ) were then radiolabelled at their 3 end by ligation of [â£-32 P]-pCp using T4 RNA ligase (New England Biolabs, Ipswich, MA, USA) according to the manufacturer's instructions (9) . NP868R recombinant protein (0.5 M) was incubated 1 h at 30 • C with 10 mM GTP and 5 M pppAC4pCp* in 50 mM Tris-HCl (pH 8.5), 5 mM DTT, 5 mM KCl su.....
Document: GTase assay. The pppAC 4 RNA was synthetized using T7 DNA primase as described by Peyrane et al. (8) . The RNA (pppAC 4 ) were then radiolabelled at their 3 end by ligation of [â£-32 P]-pCp using T4 RNA ligase (New England Biolabs, Ipswich, MA, USA) according to the manufacturer's instructions (9) . NP868R recombinant protein (0.5 M) was incubated 1 h at 30 • C with 10 mM GTP and 5 M pppAC4pCp* in 50 mM Tris-HCl (pH 8.5), 5 mM DTT, 5 mM KCl supplemented or not with 2.5 mM MgCl 2 . After incubation, the reactions were stopped by addition of an equal volume of formamide/EDTA gel-loading buffer and hydrolysis products were separated over a 20% polyacrylamide/8 M urea gel, prior to phosphorimaging.
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