(PLP) were purchased from Sigma-Aldrich. Isopropyl β-
the primers were from Genewiz (Suzhou, China). All of the enzymes
and other bioreagents were purchased from Takara (Dalian, China).
2 ketobutyric acid
Manufactured by Merck Group
Sourced in Germany, United States
2-ketobutyric acid is a chemical compound that can be used as a laboratory reagent. It is a colorless, crystalline solid that is soluble in water and organic solvents. The compound has a molecular formula of CH3CH2COCOOH.
Automatically generated - may contain errors
Lab products found in correlation
L threonine, by Merck Group (2 mentions)
Pyridoxal 5 phosphate hydrate, by Merck Group (1 mentions)
Isopropyl β d thiogalactoside, by Promega (1 mentions)
L valine, by Merck Group (1 mentions)
L isoleucine, by Merck Group (1 mentions)
Yeast nitrogen base without amino acids, by Merck Group (1 mentions)
Lb medium, by Carl Roth (1 mentions)
Synthetic complete drop out medium, by Formedium (1 mentions)
Oleamide, by Merck Group (1 mentions)
Sodium succinate dibasic hexahydrate, by Merck Group (1 mentions)
Uridine, by Merck Group (1 mentions)
Oxaloacetic acid, by Merck Group (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
β nicotinamide adenine dinucleotide hydrate, by Merck Group (1 mentions)
Dimethyl α ketoglutarate, by Merck Group (1 mentions)
L malic acid, by Merck Group (1 mentions)
L aspartic acid, by Merck Group (1 mentions)
Sodium l lactate, by Merck Group (1 mentions)
Ferrostatin 1 hy 100579, by MedChemExpress (1 mentions)
Doxycycline d9891, by Merck Group (1 mentions)
4 protocols using 2 ketobutyric acid
1
Biosynthesis of Branched-Chain Amino Acids
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l -Threonine, l -isoleucine, l -valine, 2-ketobutyric acid, and pyridoxal-5-phosphate hydrate
(PLP) were purchased from Sigma-Aldrich. Isopropyl β-d -thiogalactoside (IPTG) was supplied by Promega (Madison). All of
the primers were from Genewiz (Suzhou, China). All of the enzymes
and other bioreagents were purchased from Takara (Dalian, China).
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(PLP) were purchased from Sigma-Aldrich. Isopropyl β-
the primers were from Genewiz (Suzhou, China). All of the enzymes
and other bioreagents were purchased from Takara (Dalian, China).
2
Yeast Genetic Transformation Protocol
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l -threonine, 2-ketobutyric acid, (R)- and (S)-aminobutyric acid, (R) and (S)-2-aminobutanol were bought from Sigma-Aldrich Chemie AG (Steinheim, Germany). Yeast Extract Peptone (YPD) medium with 20 g/L glucose was used for growth of the wildtype strain prior to transformation and both bought from Sigma-Aldrich Chemie AG. Synthetic complete (SC) drop out medium (Formedium LTD, Hustanton, England) with 6.7 g/L yeast nitrogen base without amino acids and ammonium sulfate (Sigma-Aldrich Chemie AG), and 20 g/L glucose (Sigma-Aldrich Chemie AG) was used for pre- and main cultures. LB medium for growth of E. coli was supplied from Carl Roth GmbH + Co. KG (Karlsruhe, Germany).
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3
Metabolite supplementation experiments
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In certain experiments (one of) the following compounds was/were added to the medium: sodium pyruvate (#11360070, Life Technologies, Carlsbad, CA, USA), l -aspartic acid (#A7219, Sigma-Aldrich), 2-ketobutyric acid (#K401, Sigma-Aldrich), oxaloacetic acid (#O4126, Sigma-Aldrich), sodium l -lactate (#L7022, Sigma-Aldrich), l -(-)-malic acid (#M1000, Sigma-Aldrich), sodium succinate dibasic hexahydrate (#S9637, Sigma-Aldrich), dimethyl α-ketoglutarate (#349631, Sigma-Aldrich), uridine (#U3003, Sigma-Aldrich), β-nicotinamide adenine dinucleotide hydrate (#43410, Sigma-Aldrich), Oleamide (#O2136, Sigma-Aldrich) These compounds were dissolved directly in the medium after which its pH was adjusted to 7.2 with NaOH. Media were freshly prepared prior to each experiment.
4
Cell Culture and Hypoxia Induction
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HeLa (CCL-2), MDA-MB-231 (HTB-26), and HEK293T (CRL-3216) cell lines were obtained from ATCC and were maintained in DMEM (Gibco, C11995500BT) with 10% fetal bovine serum (Gibco, 10099141 C) and 1% penicillin-streptomycin (Gibco, 15140-122) at 37 °C within a humidified atmosphere of 20% O2 and 5% CO2. Primary embryonic fibroblasts (MEFs) were prepared from E13.5 wild type C57BL/6 embryos using standard protocols35 and maintained in DMEM with 10% fetal bovine serum. MEFs, cultured for no more than six passages, were used for experiments. Since pyruvate could attenuate the accumulation of NADH, we replaced the medium with pyruvate-free DMEM (Merck, D0819) in NADH-related experiments to avoid the interference of pyruvate. To induce hypoxia, cells were cultured under 0.5% oxygen conditions. All the cell lines were tested negative for mycoplasma, and no commonly misidentified cell lines were used in the study. Doxycycline (D9891), N-acetyl-L-cysteine (A9165), glutathione monoethyl ester (353905), and 2-ketobutyric acid (K401) were purchased from Sigma. 6-Mercaptopurine (HY-13677), pelitrexol (HY-14530), lometrexol (HY-14521B), Z-VAD-FMK (HY-16658B), necrosulfonamide (HY-100573), ferrostatin-1 (HY-100579), and menadione (HY-B0332) were purchased from MedChemExpress. A769662 (S2697) and AICAR (S1802) were purchased from Selleck.
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