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Bovine erythrocyte sod

Manufactured by Merck Group
Sourced in United States

Bovine erythrocyte SOD is a laboratory product that contains the enzyme superoxide dismutase (SOD) isolated from bovine erythrocytes. SOD is an antioxidant enzyme that catalyzes the conversion of superoxide radicals into oxygen and hydrogen peroxide.

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6 protocols using bovine erythrocyte sod

1

Oxidative Stress Assays in Hippocampus

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Hippocampal SOD activity was determined using an SOD assay kit (Dojindo Laboratories; Kumamoto, Japan). Absorbance was measured at 450 nm using a UV spectrophotometer, and the results were calculated using dilutions of bovine erythrocyte SOD (Sigma) ranging from 0.01–50 units/mL.
Hippocampal catalase activity was analyzed as previously described [30 (link)]. The hippocampal homogenates were mixed with phosphate buffer (250 mM, pH 7.0), methanol (12 mM), and H2O2 (44 mM) in sequence. After incubation at RT for 20 min, the reaction was stopped by the addition of Purpald solution (22.8 mM Purpald in 2 N potassium hydroxide). The mixture was incubated at 25 °C for 20 min; then, potassium periodate (65.2 mM in 0.5 N potassium hydrate) was added. Absorbance at 550 nm was measured using a UV spectrophotometer, and the results were calculated using a catalase standard curve.
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2

Measurement of Hepatic Antioxidant Enzymes

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SOD activity in hepatic tissue was determined using a SOD assay kit (Dojindo Laboratories, Kumamoto, Japan), according to the manufacturer's protocol. Bovine erythrocyte SOD (Sigma, MO, USA) was used as a standard. 20 μL of standard or diluted hepatic tissue homogenate was mixed with 200 μL of WST-1 working solution in a 96-well plate. Subsequently, 20 μL of enzyme working solution was added to each well and thoroughly mixed. The plate was incubated at 37°C for 20 min, and 450 nm was measured using a microplate reader (Molecular Devices).
CAT activity in the hepatic tissue was determined using the method of the previously described method [26 (link)]. Briefly, 140 μL of phosphate buffer (250 mM, pH 7.0), 150 μL of 12 mM methanol, and 30 μL of H2O2 were mixed with 300 μL of standard or diluted hepatic tissue homogenate in a 13 × 100 mm test tube. The reaction was allowed to proceed for 10 to 20 min and was stopped by the addition of 450 μL of Purpald (22.8 mM in 2 N potassium hydroxide). The mixture was maintained for 20 min at 25°C, and then 150 μL of potassium periodate (65.2 mM in 0.5 N potassium hydrate) was added to the same tube. The absorbance was measured at 550 nm using a microplate reader (Molecular Devices).
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3

Hepatic Antioxidant Enzyme Assays

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Superoxide dismutase (SOD) activity in hepatic tissue was determined using a SOD assay kit (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer' protocol. Bovine erythrocyte SOD (Sigma) was used a standard. 20 μL of standard or diluted hepatic tissue homogenate were mixed with 200 μL of WST-1 working solution in a 96-well plate. Subsequently, 20 μL of enzyme working solution were added to each well and thoroughly mixed. The plate was incubated at 37°C for 20 min, and was measured at 450 nm using a microplate reader (Molecular Device Corp., Sunnyvale, CA, USA).
Catalase activity in the hepatic tissue was determined according to the previous method [26] (link). The protein sample reacted with hydrogen and Purpald solution (22.8mM of Purpald in 2N potassium hydroxide). The absorbance at 550 nm of the purple formaldehyde adduct was measured using a spectrophotometer.
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4

Enzymatic Activities Measurement Protocol

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SOD activity was determined using an SOD assay kit according to the manufacturer's protocol (Dojindo Laboratories, Kumamoto, Japan). The standard concentration was serially diluted from 100 to 0.01 U/mL of bovine erythrocyte SOD (Sigma Aldrich).
Catalase activity was determined as previously described [20 (link)]. The absorbance of the purple formaldehyde adduct was measured at 550 nm using a spectrophotometer (Molecular Devices Corp.).
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5

Hippocampal Oxidative Stress and Antioxidants

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We obtained hippocampal homogenates from stored hippocampal tissue (n = 6 to 9 in each group) to verify parameters for oxidative stress and analyze antioxidant components. We used commercial kits for all assays, and all measurements were taken according to the manufacturer’s instructions. The degree of lipid peroxidation was measured by the level of malondialdehyde (MDA) using Lipid Peroxidation Colorimetric/Fluorometric Assay Kit (Catalog # K739, BioVision Inc., Milpitas, CA, USA). The assay’s final products were analyzed and quantified using a spectrophotometer (SoftMax, Ver. 5.4, Molecular Devices, Sunnyvale, CA, USA) at a wavelength of 530 nm. Total GSH content was determined using GSH Assay Kit (Catalog # CS0260, Sigma-Millipore) and the absorbance was calculated at a wavelength of 405 nm using the spectrophotometer (VersaMax. Ver. 5.1, SoftMax). Superoxide dismutase (SOD) activities were measured using a commercially available kit. As standards, dilutions of bovine erythrocyte Sod (Sigma-Aldrich, St. Louis, MO, USA) in concentrations ranging from 0.01 to 50 U/mL were used. Amplex™ Red Catalase Assay Kit (Catalog # A22180, Thermo Fishers, Waltham, MA, USA) was used to measure catalase activities. The absorbance was measured at a wavelength of 450 nm using a plate reader.
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6

Cerebral Cortex and Hippocampus Catalase and SOD Assays

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Catalase activities in cerebral cortex and hippocampus were assayed as described previously [24] (link). Briefly, 150 µL of phosphatase buffer (250 mM, pH 7.2),150 µL of 12 mM methanol and 30 mL of 44 mM H2O2 were mixed with 300 µL of each homogenate sample/standard solutions in a 13×100 mm test tube. The reaction was allowed to proceed for 10 to 20 min and was stopped by the addition of 450 mL of Purpald solution (22.8 mM of Purpald in 2 N potassiumhydroxide). The mixture was kept for 20 min at 25°C, followed by addition of 150 µL of potassium periodate (65.2 mM in 0.5 N potassiumhydrate). The absorbance of the purple formaldehyde adduct formed was measured at 550 nm using a spectrophotometer (Molecular Devices, Sunnyvale, CA).
SOD activity in cerebral cortex and hippocampus was determined using a SOD assay kit (Dojindo Laboratories, Kumamoto, Japan), according to the protocol from the manufacture. Bovine erythrocyte SOD (Sigma) was used as a standard (0.01 - 20 U/mL).
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