Glutathione reductase (GR) from baker’s yeast, Urease, glucose oxidase (GOx), trypsin, and acetaminophen (APAP) were purchased from Sigma-Aldrich (Shanghai, China). Reduced glutathione (GSH), oxidized glutathione (GSSG), hemin, bovine serum albumin (BSA), and curcumin (CMN) were obtained from Aladdin Chemical Reagent Co., Ltd. (Shanghai, China). Urea, folic acid (FA), glucose, arginine, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), and β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH) were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The stock solution of 5.0 mM hemin was prepared in dimethyl sulfoxide (DMSO) and stored in darkness at −20 °C. All other reagents were of analytical grade and used as received without further purification. Ultrapure water with a resistivity of 18.2 MΩ cm obtained from a Milli-Q purification system was used throughout the experiments. Oligonucleotide (5′-TTT GGG TAG GGC GGG TTG GG-3′, G4-DNA) was synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).
Glutathione reductase from baker s yeast
Manufactured by Merck Group
Sourced in United States, Germany
Glutathione reductase from baker's yeast is an enzyme that catalyzes the reduction of glutathione disulfide to the sulfhydryl form glutathione, which is an important antioxidant. The enzyme is isolated from the yeast Saccharomyces cerevisiae.
Automatically generated - may contain errors
Lab products found in correlation
Hydrochloric acid (hcl), by Merck Group (2 mentions)
Sodium azide, by Merck Group (2 mentions)
Urease, by Merck Group (1 mentions)
Oligonucleotide, by Sangon (1 mentions)
Urea, by Sangon (1 mentions)
Potassium cyanide, by Merck Group (1 mentions)
Xanthine oxidase from bovine milk, by Merck Group (1 mentions)
Trichloroacetic acid, by Kemika (1 mentions)
Butylated hydroxy toluene, by Thermo Fisher Scientific (1 mentions)
Absolute ethanol, by ITW Reagents (1 mentions)
Ethylenediaminetetraacetic acid tetrasodium salt, by Merck Group (1 mentions)
Superoxide dismutase sod from bovine erythrocytes, by Merck Group (1 mentions)
2 thiobarbituric acid, by Thermo Fisher Scientific (1 mentions)
Ethylenediaminetetraacetic acid disodium salt edta 2na, by GE Healthcare (1 mentions)
Glutathione (gsh), by Merck Group (1 mentions)
Dmdse, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Nadph, by Merck Group (1 mentions)
Lambda 2 spectrophotometer, by PerkinElmer (1 mentions)
Mercaptoethanol, by Merck Group (1 mentions)
8 protocols using glutathione reductase from baker s yeast
1
Enzyme-based Assays and Biomolecule Interactions
2
Glutathione Reductase Assay Protocol
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Glutathione reductase (GR) from baker's yeast (S. cerevisiae) (100-300 U mg -1 protein), superoxide dismutase (SOD) from bovine erythrocytes (3000 U mg -1 protein), xanthine oxidase from bovine milk (0.4-1.0 U mg -1 protein), ethylenediaminetetraacetic acid tetrasodium salt, nitrotetrazolium blue chloride (NBT), xanthine, and potassium cyanide were purchased from Sigma Aldrich (Germany). A stabilised 3 % solution of hydrogen peroxide was purchased from Fluka (Germany), β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt ( N A D P H ) f r o m S e r v a ( G e r m a n y ) , a n d ethylenediaminetetraacetic acid disodium salt (EDTA-2Na) from Pharmacia Biotech (Sweden). Acetonitrile and dimethyl sulfoxide (DMSO) were purchased from J.T. Baker (Italy), yeast extract (YES) from Biolife (Italy), while aflatoxin standard mix (AFB1, AFG1, AFB2, and AFG2) from Biopure (Austria). Sodium azide and hydrochloric acid were purchased from Merck (Germany), trichloroacetic acid from Kemika (Croatia), ethanol absolute from Panreac (Spain), and butylated hydroxytoluene and 2-thiobarbituric acid were purchased from Acros Organics (USA). All other chemicals were of p.a. quality and purchased from commercial suppliers.
3
Thioredoxin System Assay Protocol
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MSeA was purchased from Aldrich (Saint-Louis, MO, USA); GSH, GSSG, NADPH, DTT, DMDSe, and TCEP from Sigma (Saint-Louis, MO, USA). DMDSe was prepared as a 20 mM stock solution in 100% ethanol stored at −20 °C. Glutathione reductase from baker’s yeast (205 units/mg, 2.2 mg/mL) was from Sigma (Saint-Louis, MO, USA). Yeast thioredoxin (9.7 units/mg, 88.5 µM) was from Fisher Scientific (Waltham, MA, USA). Yeast thioredoxin reductase (25.6 units/mg) was purchased from Abnova (Taipei, Taiwan).
4
Purification and Assay of Thioredoxin Reductases
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Highly
purified cytosolic (TrxR1) and mitochondrial (TrxR2) thioredoxin reductases
were prepared from rat liver according to Luthman and Holmgren93 (link) and Rigobello and Bindoli,94 (link) respectively. The protein content of the purified enzyme
preparations was measured according to Lowry et al.95 (link) Glutathione reductase from baker’s
yeast was purchased from Sigma-Aldrich (St. Louis, MO, USA). Thioredoxin
reductase activity was determined by estimating the DTNB reducing
properties of the enzymes in the presence of NADPH. Aliquots of highly
purified TrxR in 0.2 M Na–K–Pi buffer (pH 7.4) added
to 5 mM EDTA were pre-reduced with 0.25 mM NADPH. Then, the compounds
were added at different concentrations and the reaction was initiated
with 1 mM DTNB and monitored spectrophotometrically at 412 nm for
about 10 min on a Lambda 2 spectrophotometer (PerkinElmer, Waltham,
MA, USA). GR activity was measured in 0.2 M Tris–HCl buffer
(pH 8.1), 1 mM EDTA, and 0.25 mM NADPH in the presence of the compounds
at increasing concentrations. The assay was started by the addition
of 1 mM GSSG and followed spectrophotometrically at 340 nm.
purified cytosolic (TrxR1) and mitochondrial (TrxR2) thioredoxin reductases
were prepared from rat liver according to Luthman and Holmgren93 (link) and Rigobello and Bindoli,94 (link) respectively. The protein content of the purified enzyme
preparations was measured according to Lowry et al.95 (link) Glutathione reductase from baker’s
yeast was purchased from Sigma-Aldrich (St. Louis, MO, USA). Thioredoxin
reductase activity was determined by estimating the DTNB reducing
properties of the enzymes in the presence of NADPH. Aliquots of highly
purified TrxR in 0.2 M Na–K–Pi buffer (pH 7.4) added
to 5 mM EDTA were pre-reduced with 0.25 mM NADPH. Then, the compounds
were added at different concentrations and the reaction was initiated
with 1 mM DTNB and monitored spectrophotometrically at 412 nm for
about 10 min on a Lambda 2 spectrophotometer (PerkinElmer, Waltham,
MA, USA). GR activity was measured in 0.2 M Tris–HCl buffer
(pH 8.1), 1 mM EDTA, and 0.25 mM NADPH in the presence of the compounds
at increasing concentrations. The assay was started by the addition
of 1 mM GSSG and followed spectrophotometrically at 340 nm.
5
EAE Induction and Oxidative Stress Analysis
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The chemicals and drugs used for EAE induction were myelin oligodendrocyte glycoprotein peptide human fragment (MOG35–55 MEVGWYRPPFSRVVHLYRNGK), complete Freund’s adjuvant (CFA; heat-killed Mycobacterium tuberculosis 1 mg/ml) and pertussis toxin (PTX) purchased from Sigma-Aldrich (Darmstadt, Germany), ethanol and acetone purchased from Prolabo (Laboratoires humeau, La Chapelle-sur-Erdre, France), and sterile 0.9% sodium chloride (NaCl; CDM Lavoisier, Paris, France).
For biochemistry of oxidative stress and Western blotting analysis, the chemicals and drugs used were phosphate-buffered saline pH = 7.4 (PBS; Gibco, Grand Island, NY, USA), acetate and ethanol purchased from Prolabo (Laboratoireshumeau, La Chapelle-sur-Erdre, France), 2,6-di-tert-butyl-4-methylphenol (BHT), trichloroacetic acid (TCA), 2-thiobarbituric acid (TBA), 1,1,3,3-tetrathoxypropane (TMP), dinitrophenylhydrazine (DNPH), hydrochloric acid (HCl), hydrogen peroxide solution, sodium azide, ethylenediaminetetraacetic acid (EDTA), β-nicotinamide adenine dinucleotide phosphate sodium salt reduced (NADPH), SOD, CAT, glutathione reductase from baker’s yeast, reduced glutathione (GSH), GPx, bromophenol blue, and mercaptoethanol, all purchased from Sigma-Aldrich (Darmstadt, Germany).
For biochemistry of oxidative stress and Western blotting analysis, the chemicals and drugs used were phosphate-buffered saline pH = 7.4 (PBS; Gibco, Grand Island, NY, USA), acetate and ethanol purchased from Prolabo (Laboratoireshumeau, La Chapelle-sur-Erdre, France), 2,6-di-tert-butyl-4-methylphenol (BHT), trichloroacetic acid (TCA), 2-thiobarbituric acid (TBA), 1,1,3,3-tetrathoxypropane (TMP), dinitrophenylhydrazine (DNPH), hydrochloric acid (HCl), hydrogen peroxide solution, sodium azide, ethylenediaminetetraacetic acid (EDTA), β-nicotinamide adenine dinucleotide phosphate sodium salt reduced (NADPH), SOD, CAT, glutathione reductase from baker’s yeast, reduced glutathione (GSH), GPx, bromophenol blue, and mercaptoethanol, all purchased from Sigma-Aldrich (Darmstadt, Germany).
6
Mifepristone and Misoprostol Assay
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Mifepristone and misoprostol (neat oil) were purchased from Cayman Chemical (Ann Arbor, MI, United States). Urethane, Tween® 80, 5,5′-dithiobis(2-nitrobenzoic acid), 2-thiobarbituric acid (TBA), L-glutathione reduced (GSH) and glutathione reductase from baker’s yeast were purchased from Sigma-Aldrich (St. Louis, MO, United States). Carboxymethylcellulose sodium (CMC-Na), 2-vinylpyridine, NADPH, 1-chloro-2,4-dinitrobenzene and trichloroacetic acid (TCA) were purchased from VWR (Philadelphia, PA, United States). All other chemicals were of the highest grade commercially available.
7
Analytical Reagents for Enzyme Assays
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Acetonitrile (99%), amitraz (> 98.0%), coumaphos (99.0%, Pestanal™ analytical standard), 7-ethoxycoumarin, tau-fluvalinate (98.7%), 1-naphthol (> 98.0%), 1-naphthyl acetate (> 98.0%), sodium dodecyl sulfate (~ 99%), Trizma base primary standard and buffer (> 99%), glutathione reductase from Baker's yeast, 1-chloro-2,4-dinitrobenzene (≥ 99%), and fast blue RR salt (95%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetone (HPLC grade), sodium phosphate dibasic anhydrous (98.0%), sodium phosphate monobasic monohydrate (99.5%), Triton X-100, hydrochloric acid (12.1 N), and sodium chloride (≥ 99.0%) were purchased from Fisher Scientific (Hampton, NH, USA). Coomassie brilliant blue G (electrophoresis grade) was purchased from MP Biomedical Supplies LLC (Solon, OH, USA). 7-hydroxycoumarin (99%) was purchased from Acros Organics (Pittsburg and Westchester, PA, USA).
8
Quantifying Glutathione in Sel-Treated HeLa Cells
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HeLa cells (4.5 x 10 5 ) were incubated for 18 h with increasing concentrations of Sel (5-20 µM). Then, cells were washed with cold PBS, and directly deproteinized with 6% meta-phosphoric acid. After 20 min at 4°C cells were scraped and centrifuged at 15800g for 10 min at 4°C. Supernatants were neutralized with 15% Na 3 PO 4 and utilized for total glutathione estimation. 23 Aliquots of the samples were incubated with 0.2 Please do not adjust margins Please do not adjust margins mM NADPH and 0.4 units of glutathione reductase from baker's yeast (Sigma-Aldrich, St. Louis, MO, USA) in 0.2 M Na-K-Pi buffer (pH 7.4) added of 5 mM EDTA. The reaction was started by the addition of 0.25 mM DTNB and the absorbance was monitored spectrophotometrically at 412 nm for about 10 min. The nanomoles of total glutathione were calculated using a standard curve of glutathione. For measurement of oxidized glutathione, sample aliquots were treated with 2% 2vinylpyridine for 40 min before performing the assay. 24 For protein estimation, cell pellets were washed with 1 mL of icecold acetone, centrifuged at 11000g, dried, then dissolved in 62.5 mM Tris-HCl buffer (pH 8.1) containing 1% SDS and analyzed by the Lowry assay. 25
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