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3 protocols using β nicotinamide adenine dinucleotide reduced form

1

Antioxidant Evaluation in Diabetic Rat Model

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β-Nicotinamide adenine dinucleotide-reduced form (NADH), β-Nicotinamide adenine dinucleotide phosphate-reduced form (NADPH), 2-thiobarbituric acid (TBA), 1,1,3,3-tetraethoxy propane (TEP), DL-glyceraldehyde, lithium sulfate, β-mercaptoethanol, reduced glutathione (GSH), 5,5′-dithiobis-(2-nitrobenzoic acid; DTNB), and melatonin (MT), were obtained from Sigma-Aldrich Company (St. Louis, MO, USA). Streptozotocin (STZ) was purchased from Upjohn Company (Kalamazoo, MI, USA). Streptozotocin was reconstituted immediately before use with 9.5 ml of 0.9% NaCl and 5% dextrose according to the manufacturer’s instruction. Enzymatic kit used for determination of glucose was prepared from Pars Azemoon Company (Tehran, Iran). HbA1c kit was obtained from Intermedical Company (Villaricca, Italy). All other reagents were obtained from other commercial sources.
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2

Preparation of Biochemical Reagents

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Flavin adenine dinucleotide (FAD, disodium salt hydrate, > 95%), riboflavin 5′-monophosphate (FMN, sodium salt hydrate, 73–79% pure, < 6 % of free riboflavin and < 6% of riboflavin diphosphate), (−)-riboflavin (> 95%), adenosine 5′-monophosphate (AMP, disodium salt, >99 %), adenosine 5′-diphosphate (ADP, sodium salt, > 95 %), and β-nicotinamide adenine dinucleotide, reduced form (NADH, disodium salt, > 97 %) were purchased from Sigma-Aldrich or Merck. Doubly de-ionized water with a conductivity of less than 17.3 μS cm was used to prepare all aqueous solutions. All other commercial chemicals were analytical grade and used without further purification.
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3

Protein Purification and Enzyme Assay

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PLP, L-alanine, sodium glyoxylate, rabbit muscle L-lactic dehydrogenase (LDH), β-Nicotinamide adenine dinucleotide reduced form (NADH), isopropyl-β-D-thiogalactoside (IPTG), LCS, DCS and imidazole were all purchased from Sigma. Ham's F12 Glutamax medium and Zeocin were purchased from Invitrogen. Geneticin was purchased from Gibco. Protease Inhibitor Cocktails were purchased from Roche. Water and organic solvents were purchased from Thermo Fisher Scientific. All other chemicals unless otherwise noted, were from Sigma-Aldrich or Wako.
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