ATP, GTP, UTP, CTP, creatine phosphate, and creatine kinase were purchased from Roche Applied Science (Indianapolis, IN). Streptavidin-coated microbeads were purchased from Thermo Scientific (Rockford, IL). L-[U-14C] Leucine was from Perkin Elmer (Waltham, MA). FITC-conjugated murine anti c-myc antibody was purchased from Santa Cruz Biotechnology (Dallas, TX). Biotin X-NTA was from Anaspec (Fremont, CA). All other chemical reagents were obtained from Sigma-Aldrich (St. Louis, MO) and used without further purification. The S12 extract prepared from the Escherichia coli strain BL21 Star (DE3) (Invitrogen, Carlsbad, CA) was used as a source of protein synthesis machinery, as described previously [19 (link)]. Human antisera against C. sinensis were prepared from patients with clonorchiasis. This study involving the use of the patient sera was approved by the Research Ethics Committee of Korea National Institute of Health (approval no. 2013-06EXP-04-R).
L 14c u leucine
Manufactured by PerkinElmer
Sourced in United States
L-[14C(U)]-leucine is a radioactive amino acid product used in research and laboratory applications. It serves as a tracer to study protein synthesis, amino acid metabolism, and other biological processes. The product provides a source of radioactively-labeled leucine for scientific investigation.
Automatically generated - may contain errors
Lab products found in correlation
Bl21 star de3, by Thermo Fisher Scientific (2 mentions)
Adenosine triphosphate (atp), by Roche (2 mentions)
Creatine kinase, by Roche (2 mentions)
D u 14c glucose, by PerkinElmer (2 mentions)
D glucose, by PerkinElmer (2 mentions)
Ultima gold llt scintillation fluid, by PerkinElmer (2 mentions)
Creatine phosphate, by Roche (1 mentions)
Streptavidin coated microbeads, by Thermo Fisher Scientific (1 mentions)
Ni nta agarose resin, by Qiagen (1 mentions)
Ampicillin, by Duchefa Biochemie (1 mentions)
Luria bertani medium, by Duchefa Biochemie (1 mentions)
L leucine, by PerkinElmer (1 mentions)
Anti actin ab3280, by Abcam (1 mentions)
Purexpress in vitro protein synthesis kit, by New England Biolabs (1 mentions)
Mf membrane, by Merck Group (1 mentions)
Tri carb liquid scintillation counter, by Hewlett-Packard (1 mentions)
Ultima gold mv scintillation cocktail, by PerkinElmer (1 mentions)
35s methionine, by PerkinElmer (1 mentions)
Amino acid, by Merck Group (1 mentions)
Spermidine, by Merck Group (1 mentions)
8 protocols using l 14c u leucine
1
In Vitro Protein Synthesis Assay
2
In Vitro Protein Synthesis System
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Sesame crude oil was purchased from the market (Daejeon, Korea). Luria-Bertani (LB) medium and ampicillin were purchased from Duchefa Biochemie (Haarlem, The Netherlands). Ni-NTA agarose resin was from Qiagen (Hilden, Germany). ATP, GTP, UTP, CTP, and creatine kinase were purchased from Roche Applied Science (Indianapolis, IN). Creatine phosphate was a kind gift from Bioneer Co. (Daejeon, Korea). L-[U-14C] leucine was from Perkin Elmer (Waltham, MA). All other chemical reagents were obtained from Sigma-Aldrich (St Louis, MI) and used without further purification. The S12 extract prepared from the E. coli strain BL21Star (DE3) (Invitrogen, Carlsbad, CA) was used as a source of protein synthesis machinery as described previously [9 (link)].
3
Metabolic Substrate Uptake and Utilization
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For the measurement of substrate uptake, lipid synthesis, and CO2 production, myocytes were incubated in HBSS containing 0.3 mM l ‐leucine, 0.3 mM l ‐isoleucine, or 5 mM d‐glucose + 2 μCi/ml of labeled 14C(u)‐l ‐leucine, 14C(u)‐l ‐isoleucine, or 14C(u)‐d‐glucose (Perkin Elmer).
Uptake was measured as radioactivity incorporated in myocytes after 0, 1, 2, 3, 5, or 10 min. Lipid fractions were separated by a standard CHCl3/MeOH extraction at 0, 2, and 4 h. For CO2 production, incubation was carried out in flasks in the presence of a paper filter imbibed in 0.2 M KOH/NaOH solution. The C14‐uptake and C14‐incorporation into lipids or CO2 were measured in a scintillation counter with Ultima Gold LLT scintillation fluid (Perkin Elmer).
Uptake was measured as radioactivity incorporated in myocytes after 0, 1, 2, 3, 5, or 10 min. Lipid fractions were separated by a standard CHCl3/MeOH extraction at 0, 2, and 4 h. For CO2 production, incubation was carried out in flasks in the presence of a paper filter imbibed in 0.2 M KOH/NaOH solution. The C14‐uptake and C14‐incorporation into lipids or CO2 were measured in a scintillation counter with Ultima Gold LLT scintillation fluid (Perkin Elmer).
4
Substrate Catabolism Measurement in Myoblasts
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For the measurement of substrate catabolism, total oxidation and CO2 production were analysed in myoblasts and myocytes incubated in HBSS containing 0.3 mM l -leucine or 5 mM d -glucose + 2 μCi per ml of labelled [14C(u)]l -leucine or [14C(u)]d -glucose (Perkin Elmer). For CO2 production, incubation was carried out in flasks in the presence of a paper filter imbibed in 0.2 M KOH/NaOH solution. The [14C]CO2 activity was measured in a scintillation counter with Ultima Gold LLT scintillation fluid (Perkin Elmer).
5
Ricin Toxin Exposure Detection
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The following products were purchased from the indicated commercial sources: l -[14C(U)]-leucine was purchased from Perkin-Elmer (Waltham, MA, USA); DMSO (D4540), bafilomycin A1 (B1793), gelatin (G7765), anti-LC3B (L7543), anti-p62 (P0067) and Calcein-AM (17783) were purchased from Sigma (St. Louis, MO, USA); Alamar Blue™ Cell Viability Reagent (DAL1025) was purchased from ThermoFisher Scientific (Rockford, IL, USA); anti-actin (ab3280) was purchased from Abcam; anti-RTA (SC-52190) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Ricin toxin was a kind gift from Dr. Bruno Beaumelle, Institut de Recherche en Infectiologie de Montpellier, France.
6
Cell-Free Protein Synthesis with Liposomes
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The PURExpress® In Vitro Protein Synthesis Kit (New England Biolabs) was used for all experiments. Reactions were set up following the manufacturer’s instructions, supplemented with previously prepared liposomes (at a final lipid concentration of 3–5 mg.ml−1), and a protease inhibitor solution used to make the reaction up to the required volume. Reactions were typically 12.5, 25 or 50 µl, with 50 ng.μl−1 plasmid DNA. The reaction was then incubated at 30 °C for 2–3 hours. For reactions containing [35S]Methionine (PerkinElmer), 0.04–0.1 mCi.ml−1 was used in the cell free reaction. For reactions containing L-[14C(U)]-Leucine (PerkinElmer) 6 μCi.ml−1 was used. To measure the rate of elongation, 2 µl of the cell free reaction was removed at each time point, and pipetted onto 0.45 μm MF-Membrane (Millipore). The protein made was quantified according to the PURExpress® In Vitro Protein Synthesis Kit manual and Wuu and Swartz56 (link), with a 1600 TR Tri-Carb® Liquid Scintillation Counter (Packard) and using the Ultima Gold MV scintillation cocktail (PerkinElmer).
7
E. coli CFPS of GRFT Optimization
8
Radiolabeled Amino Acid Transport Assay
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l -Cysteine hydrochloride monohydrate, l -cystine dihydrochloride, and other nonradioactive amino acids were purchased from Sigma-Aldrich. A 30% (wt/wt) solution of hydrogen peroxide (H2O2), bovine catalase, ovalbumin, O-nitrophenol-β-galactoside (ONPG), diethylenetriamine pentaacetic acid (DTPA), dithiothreitol (DTT), 2,2′-dipyridyl, β-mercaptoethanol, antibiotics, and 5-bromo-4-chloro-3-indolyl-β-d -galactopyranoside (X-Gal) were purchased from Sigma-Aldrich. Coomassie reagent was from Thermo Scientific. Medium reagents and buffer chemicals were from Fisher Chemical. Restriction and ligation enzymes were from New England BioLabs. Qiagen kits were used for genomic and plasmid DNA preparation. Colony PCR beads were from GE Healthcare, and PCR reagents were purchased from Invitrogen. DNA sequencing was performed at ACGT, Inc. Radiolabeled l -[1,2,1′,2′-14C]cystine and l -[14C(U)]leucine were purchased from Perkin Elmer; we note that putative radiolabeled cystine that was received from another supplier was found not to be authentic cystine. Transport measurement filters were purchased from Millipore Sigma (GSWP02500).
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