In vitro translation extracts were made from human 293T cells as described23 (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.
Amino acids
Manufactured by Promega
Amino acids are the fundamental building blocks of proteins. They are organic compounds that consist of a central carbon atom bonded to an amino group, a carboxyl group, and a side chain. Amino acids are essential for various biological processes, including protein synthesis, enzymatic reactions, and cellular signaling.
Automatically generated - may contain errors
Lab products found in correlation
Creatine phosphate, by Roche (4 mentions)
Creatine phosphokinase, by Roche (3 mentions)
Murine rnase inhibitor, by Promega (2 mentions)
Sw40ti rotor, by Beckman Coulter (1 mentions)
Hepes koh, by Promega (1 mentions)
Murine rnase inhibitor, by New England Biolabs (1 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Tristar lb 941 luminometer, by Berthold Technologies (1 mentions)
Renilla luciferase assay system, by Promega (1 mentions)
Nanoluc luciferase assay kit, by Promega (1 mentions)
Mg oac 2, by Promega (1 mentions)
Spark multimode microplate reader, by Tecan (1 mentions)
Creatine kinase, by Roche (1 mentions)
Luciferase assay reagent, by Promega (1 mentions)
6 protocols using amino acids
1
Ribosome Initiation Complex Purification
2
In vitro translation of Renilla luciferase mRNA
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Capped mRNAs coding for Renilla luciferase (Luc R) were produced by a T7 Message Machine Kit system (Ambion, Life Technologies, Saint Aubin, France) from pGb-Eg2-410Δ2-hxG-A65 EcoRV-linearized vector.33 (link) RNA was then purified by phenol-chloroform extraction and controlled by gel electrophoresis. In vitro translation was carried out in the Flexi-rabbit reticulocyte system (RRL, Promega), supplemented with 20 mM amino acids (Promega), 1 U/μl RNase inhibitor (Ambion), potassium chloride (KCl), and 5 fmol mRNA per reaction. Renilla activity from translation reaction was measured using the Renilla Luciferase Assay System (Promega) on a Tristar LB941 luminometer (Berthold, Thoiry, France).
3
In vitro Translation Assay with Nuclease-treated Lysates
4
In vitro Translation Assay with SARS-CoV-2 nsp1
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In vitro translation reactions were performed using HEK293T translation-competent cell lysate, as previously described, with modifications (Lee et al., 2015 (link)). Translation reactions contained 50% HEK293T translation-competent cell extract, 2mM ATP, 0.42mM GTP, 7mM tris(2-carboxyethyl)phosphine, 28mM HEPES pH 7.5, 2mM creatine phosphate (Roche), 0.01 μg/μl creatine kinase (Roche), 2mM Mg(OAc)2, 60mM KOAc, 10μM amino acids (Promega), 0.21mM spermidine, 0.6mM putrescine and 0.8U/ml murine RNase inhibitor (NEB). Translation reactions were pre-incubated with the corresponding recombinant nsp1 variant for 10 min at 4°C before addition of the mRNA. 40nM of the corresponding RNA was then added to the reaction. Translation reactions were then incubated at 30°C for 30 min. Luciferase assays were then performed using the NanoLuc luciferase assay kit (Promega), following the manufacturer’s protocol. Luminescence was measured using the Spark multimode microplate reader (TECAN). Technical triplicate measurements were taken for each biological replicate. These technical triplicates were averaged to plot for a given biological replicate and normalized to independent luminescence measurements from in vitro translation reactions containing the corresponding concentration of GST in place of nsp1.
5
In vitro Translation Assay with Nuclease-treated Lysates
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In vitro translation extracts were made from human 293T cells as previously described21 (link). Lysates were nuclease-treated with 18 gel units μl-1 micrococcal nuclease (NEB) 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 and buffer to make the final reaction with 0.84 mM ATP, 0.21 mM GTP, 21 mM creatine phosphate (Roche), 45 U ml-1 creatine phosphokinase (Roche), 10 mM HEPES-KOH pH 7.6, 2 mM DTT, 8 mM amino acids (Promega), 255 mM spermidine, 1 U ml-1 murine RNase inhibitor (NEB), and mRNA-specific concentrations of Mg(OAc)2 and KOAc. The optimal magnesium and potassium levels to add were determined to be 1.5 mM Mg(OAc)2 and 150 mM KOAc for c-Jun mRNA, and 1 mM and 150 mM KOAc for ACTB mRNA. For luciferase assays, translation reactions were incubated for 1 h at 30 °C, then luciferase activity was assayed.
6
Staphylococcal and E. coli Cell-free Assays
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Staphylococcal S30 extract was prepared from S. aureus RN4220 following the protocol of Murray et al. (44 (link)), although the preincubation step was omitted. E. coli S30 extract was from Promega (Madison, WI). For T/T assays, an optimized quantity of S30 extract was added into a 25-µl reaction mixture containing 0.1 mM amino acids (Promega), 10 µl S30 premix (Promega), 1 µg of DNA template (pSAluc or pBESTluc for S. aureus and E. coli T/T reactions, respectively), and antibiotics and purified protein as required. Reaction mixtures were incubated for 1 h at 37°C, and the level of transcription/translation was quantified by monitoring the expression of luciferase produced from pSAluc/pBESTluc by the addition of luciferase assay reagent (Promega) and measurement of luminescence.
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