The molecular weight (MW) distribution of HB-control and Al-HB were determined by gel filtration chromatography on a Superdex Peptide Increase 10/300 GL size exclusion column with separation range of between 100 to 700 Da (10 × 300 mm, GE Healthcare, Chicago, IL, USA) connected to a fast protein liquid chromatography (FPLC, AKTApure, GE Healthcare, Chicago, IL, USA) equipped with a UV detector at 220 nm [28 (link)]. The HB-control and Al-HB were separately dissolved in 20 mM sodium phosphate buffer containing 0.1 M NaCl (pH 7). The mixture was filtered, and the filtrate (0.5 mL) was loaded onto the column that had been pre-equilibrated with 20 mM of sodium phosphate buffer containing 0.1 M NaCl (pH 7). The elution was performed using the same buffer at a flow rate of 0.5 mL/min. The eluted peaks were monitored at 220 nm. Carbonic anhydrous (29,000 Da), aprotinin (6512 Da), glutathione oxidised (612 Da), glutathione reduced (307 Da), and glycine (75 Da) (Sigma-Aldrich, St. Louis, MO, USA) were used as MW standards. The MW distributions of HB-control and Al-HB were estimated from the linear plot of logarithm of MW against retention time.
Oxidised glutathione
Manufactured by Merck Group
Sourced in United States
Oxidised glutathione is a laboratory reagent used in biochemical and cell biology research. It serves as an important antioxidant and redox molecule in various cellular processes. The core function of oxidised glutathione is to participate in the regulation of the cellular redox state and maintain the balance between oxidative and reductive conditions within cells.
Automatically generated - may contain errors
Lab products found in correlation
Reduced glutathione, by Merck Group (5 mentions)
Glutathione reductase, by Merck Group (2 mentions)
Akta pure, by GE Healthcare (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Glycine, by Merck Group (1 mentions)
Dipalmitoylphosphatidylcholine, by Merck Group (1 mentions)
O dianisidine, by Merck Group (1 mentions)
Xanthine, by Merck Group (1 mentions)
Xanthine oxidase, by Merck Group (1 mentions)
Anti 4 hydroxynonenal antibody ab46545, by Abcam (1 mentions)
Triolein, by Merck Group (1 mentions)
Anti beta actin antibody ab8227, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Streptozotocin (stz), by Merck Group (1 mentions)
Glucose oxidase, by Merck Group (1 mentions)
Cytochrome c, by Merck Group (1 mentions)
Cholesterol, by Merck Group (1 mentions)
Horseradish peroxidase hrp, by Merck Group (1 mentions)
O phthalaldehyde, by Merck Group (1 mentions)
Nicotinamide adenine dinucleotide phosphate, by Merck Group (1 mentions)
7 protocols using oxidised glutathione
1
Molecular Weight Analysis of Hydrolyzed Biopolymers
2
Comprehensive Biochemical Assessment Protocol
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Streptozotocin, glucose oxidase, o-dianisidine, horse radish peroxidase (HRP), bovine serum albumin, cholesterol, dipalmitoylphosphatidylcholine, triolein, glutathione reductase (GR), glutathione reduced, glutathione oxidised, t-butyl hydroxyperoxide, xanthine oxidase, xanthine, cytochrome C and o-phthalaldehyde were procured from Sigma-Aldrich Chemical Co. 1-Chloro-2,4-dinitrobenzoic acid, NADP (reduced form (NADPH) and Bernhardt-Tommarelli modified salt mixture were procured from SISCO Research Laboratories. Casein was obtained from Nimesh Corporation. Maize starch, cane sugar powder and refined groundnut oil were purchased from the local market. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), LDH and creatine phosphokinase-MB (CK-MB) kits were purchased from Agappe Diagnostics Ltd. All primers (listed in Table 1) used for PCR amplifications were procured from Sigma-Aldrich Chemical Co., and antibodies (Anti-beta Actin antibody-ab8227 and Anti-4-hydroxynonenal antibody-ab46545) were from Abcam. All other chemicals and solvents used in this study were of analytical grade.
3
Alloxan-Induced Antioxidant Regulation
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Alloxan, Zingerone, reduced glutathione, nicotinamide adenine dinucleotide phosphate, glutathione reductase, oxidised glutathione were procured from Sigma Aldrich chemicals Pvt. Ltd. (USA). Rest of the chemicals used in the current study were of analytical grade.
4
Antioxidant Screening and Characterization
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2.2-diphenyl-1-picrylhydrazyl (DPPH), 2.2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS), N,N-dimethyl1,4-diaminobenzene (DMPD), reduced glutathione, oxidised glutathione, and other chemicals were purchased from Sigma-Aldrich unless otherwise stated. SeNPs (99.99% purity, size: <100 nm) were purchased from Nanografi (Ankara, Turkey). The reagents were freshly prepared for each day and used immediately. Stock standard solutions (1 mg/mL) were prepared with ACS water (Sigma-Aldrich, Prag, Czech Republic) and stored at −20 °C in the dark. Working standard solutions were prepared daily by diluting the stock solutions. The pH value was measured using WTW inoLab Level 3 with terminal Level 3 (WTW GmbH, Weilheim, Germany).
5
HLA-peptide Complex Production and Purification
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The HLA-B*57:01, HLA-B*57:03, HLA-B*58:01 and β2m genes were sub-cloned into the pET-30 expression vector and were expressed into inclusion bodies separately in Escherichia coli. The HLA complexes were refolded in the presence of the peptides listed in Table 1 and purified as described previously63 (link). Briefly, 90 mg HLA heavy chain was refolded by rapid dilution in a solution containing 3 M urea (Sigma-Aldrich, USA), 100 mM Tris-HCl, pH 8.0 (Sigma-Aldrich, USA), 400 mM l -arginine-HCl, 5 mM reduced glutathione (Sigma-Aldrich, USA) and 0.5 mM oxidised glutathione (Sigma-Aldrich, USA) in the presence of 30 mg β2m and 10 mg of the appropriate peptide for 48 h. The refolded HLA-peptide complexes were dialysed into 10 mM Tris, pH 8.0, and purified by size-exclusion chromatography using HiLoad 16/60 Superdex 200 pg (GE Healthcare, USA) columns on an AKTA Purifier (GE Healthcare, USA) FPLC chromatography systems in 10 mM Tris, pH 8.0, and 150 mM NaCl buffer. Final purification was by anion exchange using a HiTrap Q Fast Flow column (GE Healthcare, USA) on the same AKTA system in 10 mM Tris pH 8.0 buffer with a NaCl gradient from 0 to 500 mM over 45 min.
6
Characterization of Nanoparticle Structures
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Methanol, trifluoroacetic acid (TFA), sodium selenite, poly(vinyl alcohol) (PVA 49 kDa or PVA 100 kDa), reduced glutathione (GSH), and oxidised glutathione (GSSG) were obtained from Sigma-Aldrich (St. Louis, MO, USA) in ACS purity, unless noted otherwise. Deionised water underwent demineralisation by reverse osmosis using the instrument Aqua Osmotic 02 (AquaOsmotic, Tisnov, Czech Republic), and was subsequently purified using Millipore RG (18 MΏ; Millipore Corp., Billerica, MA, USA) to gain MilliQ water. The average particle size distribution was determined by quasi-elastic laser light scattering using a Malvern Zetasizer (NANO-ZS; Malvern Instruments Ltd., Worcestershire, UK). Solutions of nanoparticles were measured according to experimental conditions stated in (Dostalova et al., 2016 (link)). The structures of nanoparticles were observed using scanning electron microscopy (FE Tescan Mira II LMU, Brno, Czech Republic) under the conditions employed in (Dostalova et al., 2016 (link); Chudobova et al., 2014 (link)). Characterisation of nanoparticles is shown in Fig. 1 .
7
Oxidative Stress Biomarker Assays
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Oxidised glutathione (GSSG), 6-phosphogluconate (6-PG), glucose-6-phosphate (G6P), nicotinamide adenine dinucleotide phosphate (NADP + ), reduced nicotinamide adenine dinucleotide phosphate (NADPH + H + ), magnesium chloride (MgCl 2 ), butylparaben (butyl 4-hydroxybenzoate, >99 % purity), sodium phosphate monobasic and dibasic, tris, glutathione reductase (GR), hydrogen peroxide (H 2 O 2 ), ethylenediaminetetraacetic acid (EDTA), cOmplete™ Protease Inhibitor Cocktail, and sodium azide were obtained from Sigma-Aldrich (St. Louis, MO, USA). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea, albumin, triglyceride, creatinine, and glucose kits for biochemical analysis were obtained from Audit Diagnostics (Cork, Ireland).
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