Protein synthesis was conducted in coupled transcription/translation reactions in a final volume of 25–80 μl. Cell-free synthesis reactions were composed of 40% (v/v) translationally active CHO lysate supplemented with HEPES-KOH (pH 7.6, f.c. 30 mM, Carl Roth GmbH), sodium acetate (f.c. 100 mM, Merck), Mg(OAc)2 (f.c. 3.9 mM, Merck), KOAc (f.c. 150 mM, Merck), amino acids (complete 100 μM, Merck), spermidin (f.c. 0.25 mM; Roche), Dithiothreitol (DTT, 2.5 mM, Life technologies GmbH) and energy regenerating components including creatine phosphokinase (f.c. 0.1 mg/ml, Roche), creatine phosphate (20 mM, Roche), ATP (1.75 mM, Roche) and GTP (0.3 mM, Roche). To allow for DNA transcription during cell-free protein synthesis, 1 U/μl T7 RNA polymerase, 0.3 mM of UTP (Roche) and CTP (Roche) and 0.1 mM of the cap analogue m7G(ppp)G (Prof. Edward Darzynkiewicz, Warsaw University, Poland) were added to the reaction. Additionally, PolyG primer (f.c. 12 µM, IBA) was supplemented. To monitor the protein quantity and quality, cell-free protein synthesis reactions were supplemented with radioactive 14C-leucine (f.c. 50 μM, specific radioactivity 66.67 dpm/pmol, Perkin Elmer). Batch synthesis reactions were incubated at 30°C for 3 h at 500 rpm (Thermomixer comfort, Eppendorf).
Creatine phosphokinase
Manufactured by Roche
Creatine phosphokinase (CPK) is an enzyme found in various tissues, particularly in the heart, brain, and skeletal muscles. It plays a crucial role in the conversion of creatine to phosphocreatine, which serves as an energy storage molecule in cells. The measurement of CPK levels can provide valuable information about the health and function of these tissues.
Automatically generated - may contain errors
Lab products found in correlation
Creatine phosphate, by Roche (6 mentions)
Amino acids, by Promega (3 mentions)
Amino acid, by Merck Group (2 mentions)
Adenosine triphosphate (atp), by Roche (2 mentions)
Hepes koh, by Carl Roth (2 mentions)
Sodium acetate, by Merck Group (2 mentions)
Murine rnase inhibitor, by Promega (2 mentions)
T7 rna polymerase, by Roche (1 mentions)
Dithiothreitol dtt, by Thermo Fisher Scientific (1 mentions)
Thermomixer comfort, by Eppendorf (1 mentions)
Sw40ti rotor, by Beckman Coulter (1 mentions)
Hepes koh, by Promega (1 mentions)
Murine rnase inhibitor, by New England Biolabs (1 mentions)
Potassium acetate, by Merck Group (1 mentions)
Magnesium acetate, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
T7 rna polymerase, by Agilent Technologies (1 mentions)
6 protocols using creatine phosphokinase
1
Cell-Free Protein Synthesis Protocol
2
Ribosome Initiation Complex Purification
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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.
3
In vitro Translation Assay with S. cerevisiae S30 Extract
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For in vitro translation, an S30 extract from S. cerevisiae was prepared (see Supplemental Experimental Procedures for details) (Hofbauer et al., 1982 (link)). For in vitro translation, 2.5 μl creatine phosphokinase (10 mg/ml; Roche), 7.5 μl CaCl2 (20 mM), 25 μl 10 × translation cocktail (100 mM HEPES/KOH [pH 7.5], 10 mM Mg[OAc]2, 760 mM KCl, 4 mM GTP, 10 mM ATP, 19 amino acid mix [500 μM each; methionine excluded]), 5 μl creatine phosphate (0.6 M; Roche), and 2.5 μl Mg(OAc)2 (100 mM) were mixed with 150 μl S30 and 16 μl 35S-methionine (1,000 Ci/mmol, 10 mCi/ml). The resulting translation mix was aliquoted into 12 μl portions and filled up to 15 μl with sterile H2O, synthetic 18-mer RNA (10–200 pmol) or cycloheximide (7.5 μg/ μl), incubated at 23°C for 30 min, and the products were separated by SDS-PAGE. To test whether translation initiation or elongation are inhibited by the 18-mer RNA, complete reactions, but lacking 35S-methionine and the 18-mer RNA, were assembled and preincubated for 10 min at 23°C. Subsequently the initiated samples were cooled down to 0°C on ice for 5 min followed by the addition of 35S-methionine and 13 μM of the 18-mer RNA. Translation elongation was then carried out for 30 min at 0°C, stopped by TCA precipitation, and the products were quantified by liquid scintillation counting (see Supplemental Experimental Procedures for details).
4
Cell-Free Protein Synthesis with CHO and Sf21 Lysates
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Cell-free protein synthesis was performed using 1.5 ml reaction tubes in 25 μl in the presence of 40% CHO cell lysates, 30 mM HEPES-KOH (pH 7.5, Carl Roth GmbH), 100 mM sodium acetate (Merck), 3.9 mM magnesium acetate (Merck), 150 mM potassium acetate (Merck), 100 μM amino acids (Merck), 250 μM spermidine (Roche), 2.5 mM Dithiothreitol (Life technologies GmbH), 100 μg/ml creatine phosphokinase (Roche), 20 mM creatine phosphate (Roche), 1.75 mM ATP (Roche), 0.3 mM of UTP (Roche), 0.3 mM CTP (Roche), 0.3 mM GTP (Roche), 0.1 mM of the cap analogue m7G (pp)G (Prof. Edward Darzynkiewicz, Warsaw University, Poland) and 10 μM PolyG. For cap-independent cell-free reactions 60 ng/μl plasmid DNA was used, while 20 ng/μl purified PCR product was added to cap-dependent reactions. Moreover, 1 U/μl T7 RNA polymerase (Agilent) was added to the cell-free reaction unless otherwise stated. The cell-free reaction was incubated for 3 h at 30 °C and 600 rpm. Cell-free protein synthesis based on Sf21 cell lysates was performed equivalent to CHO based cell-free reactions.
5
In vitro Translation Assay with Nuclease-treated Lysates
6
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.
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