Superoxide Dismutase Activity Assay Kit (Colorimetric)

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Functional Studies - Superoxide Dismutase Activity Assay Kit (Colorimetric) (AB65354)

Superoxidase dismutase (ab90040) measured showing inhibition rate (%) per concentration (microgram per mL)

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PubMed

Functional Studies - Superoxide Dismutase Activity Assay Kit (Colorimetric) (AB65354)

Park J et al investigates the recovery in erectile function after administration of chronic statin alone in DM (streptozotocin (STZ)-induced diabetes mellitus) rats. SOD activity was determined using Superoxide Dismutase activity assay kit (ab65354).

* Indicates statistical significance in comparison with DM group (P < 0.05). ^# Indicates statistical significance in comparison with the statin group (P<0.05).

Image from Park J et al., PLoS One 12(2), fig 4b. Doi: 10.1371/journal.pone.0172751 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

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Functional Studies - Superoxide Dismutase Activity Assay Kit (Colorimetric) (AB65354)

Superoxide dismutase measured in biofluids at various dilutions

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Functional Studies - Superoxide Dismutase Activity Assay Kit (Colorimetric) (AB65354)

Principle of Superoxide Dismutase Assay.

• Biochemical assay

PubMed

Biochemical assay - Superoxide Dismutase Activity Assay Kit (Colorimetric) (AB65354)

Pesti-Asboth et al used Lipid Peroxidation (MDA) Assay Kit ab118970, Vitamin C Assay Kit ab65656, Glutathione Peroxidase Assay Kit ab102530, Glutathione Reductase Assay Kit ab83461, and Superoxide Dismutase Assay Kit ab65354 to investigate changes in redox in the plasma of fast growing broiler chickens.

Changes in the redox parameters in blood plasma at Days 3, 8, 21, 32, and 42. Significant differences were determined by comparing the data from each time point with the data from the first time point. Data are expressed as the means ±8 SEMs; *p<0.05, ** p<0.005, *** p<0.001.

Lipid peroxidation was determined using a commercially available assay kit (ab118970, Abcam, Cambridge, United Kingdom). The measurements were based on the reaction between MDA and thiobarbituric acid (TBA). All reagents and standard solutions were prepared according to the manufacturer's instructions. Plasma samples (20 μl) were mixed with 500 μL of 42 mM H2SO4, 125 μL of phosphotungstic acid solution was added, and each sample was mixed by vortexing and incubated at room temperature for 5 minutes. After incubation, we centrifuged the samples at 13000 x g for 3 minutes. The pellet was collected and resuspended in 100 μL of distilled water on ice with 2 μL of butylated hydroxytoluene (BHT) (100-fold dilution). The final volume was adjusted to 200 μL with distilled water. After sample preparation, the assay was performed. The MDA-TBA adduct was generated in each sample and standard solution. The TBA reagents (600 μl) were added to the samples and standards for incubation at 95°C for 60 minutes. The reaction mixes were cooled in an ice bath for 10 minutes after incubation. The samples and standards were placed in duplicate in a 96-well microplate for analysis. The absorbance of the MDA-TBA adduct was measured at 532 nm, and the calculated concentrations are expressed in nmol/ml.

To determine the vitamin C concentration, a plasma-specific assay kit was used (ab65656, Abcam, Cambridge, United Kingdom) [34–36]. The measurements were performed according to the protocol in the manual with some modifications. The kit's supernatant assay buffer was set to pH 7.0 with NaOH (10 M). The kit provides a sensitive method for vitamin C measurement. Plasma vitamin C reduces Fe3+ to Fe2+, which shows strong absorbance that can be monitored between 545–600 nm. The reagent and standard were prepared as described in the protocol. Fifty microliters of each plasma sample, run in duplicate, was diluted two fold for measurement. The assay procedure was followed as described in the manual. The absorbance of each sample was measured at 593 nm, and the concentrations were calculated as recommended and are expressed in nmol/ml.

The activity of Glutathione peroxidase (GPx) was measured with a commercially available kit (ab102530, Abcam, Cambridge, United Kingdom). In the assay, GSSG produced after GPx oxidizes GSH during H2O2 reduction. GSSG is reduced back to GSH by GR with the aid of nicotinamide adenine dinucleotide phosphate (NADPH). The reduction of NADPH is proportional to GPx activity and can be measured colorimetrically at 340 nm. The kit reagents were dissolved as described in the manual. A standard curve was prepared as described in the kit. The activity of GPx is expressed as mU/ml.

The activity of Glutathione reductase (GR) in plasma was determined using a specific assay kit (ab83461, Abcam, Cambridge, United Kingdom). In this assay, GSH is formed from GSSG by GR; then, GSH reacts with DTNB, and a 2-nitro-5-thiobenzoate anion (TNB2-) is generated. The change in absorbance was measured at 405 nm. The kit reagents were dissolved as described in the "components and storage" section. The protocol recommends pretreating the samples. First, 5 μL of 3% H2O2 was added to 100 μL of each sample. The samples were incubated at 25°C for 5 min. Then, 5 μL of catalase was added to each sample, and we incubated the samples again at 25°C for another 5 min. After the pretreatment procedure, 50 μL of each pretreated sample was added to the sample wells. The standard curve was prepared as described in the manual, and 50 μL of diluted standard solution was added to each well. GR activity is expressed in nmol/min/mL = mU/ml.

The superoxide dismutase(SOD)inhibition rate was measured with a specific assay kit (ab65354, Abcam, Cambridge, United Kingdom). In this assay, xanthine oxidase produced superoxide anions, and the conversion of superoxide anions into hydrogen peroxide was catalyzed by SOD. Superoxide anions and the water-soluble tetrazolium salt WST-1 can react to produce a water-soluble formazan dye, the absorbance of which was detected at 450 nm. The reagents were prepared as described in the kit. First, 20 μL of Blank 1, Blank 2, Blank 3 or sample was added to a 96-well plate in duplicate. Then, WST-1 solution (200 μl) was added to each blank and sample. Twenty microliters of dilution buffer were added to the Blank 2 and Blank 3 solutions. Enzyme working solution (20 μl) was added to each sample and Blank. Then, the plate was incubated at 37°C for 20 minutes. The absorbance (A) was then measured at 450 nm.

• Biochemical assay

PubMed

Biochemical assay - Superoxide Dismutase Activity Assay Kit (Colorimetric) (AB65354)

Tran et al used Lipid Peroxidation (MDA) Assay Kit ab118970 and Superoxide Dismutase Assay Kit ab65354 to investigate the effects of Gb and Ocs extracts on antioxidant enzyme activity in the brain parenchyma of the PTZ-induced epilepsy mouse model.

The quantitative measurement of antioxidant enzymes and MDA from whole-brain tissue lysates at D31 in each group treated with various combinations of drugs. Mean (n = 3) ± SD. **P ≤ 0.01 and ***P ≤ 0.001 vs. control group; #P ≤ 0.05, ##P ≤ 0.01, and ###P ≤ 0.001 vs. the PTZ group.

Lipid peroxidation (MDA) assay kits (#ab118970) were purchased from Abcam (Cambridge, UK). The SOD Activity Assay Kit (#K335-100) was purchased from BioVision, Inc. (Waltham, MA, USA) [Biovision assay kits are now manufactured by Abcam].

• Biochemical assay

PubMed

Biochemical assay - Superoxide Dismutase Activity Assay Kit (Colorimetric) (AB65354)

Bejoy et al used Lipid Peroxidation (MDA) Assay Kit ab118970 and SOD Assay Kit ab65354 to investigate the impact of cell-free hemoglobin (CFH) on ROS in transwell kidney organoids.

Co-staining for ROS (red), DAPI to mark nuclei (blue) and the proximal tubule marker LTL (green) in human kidney organoids (n=3 organoids from three independent experiments). Scale bars : 50 μm. Malondialdehyde (MDA) assay in untreated organoids versus kidney organoids receiving the CFH treatment. Superoxide dismutase (SOD) assay for the control and CFH-treated organoid groups (n=3). Bars show median values. ns, not significant; *p<0.05; **p<0.01; ****P≤0.0001 (unpaired two tailed t-test.

Free radicals present in cell culture can increase peroxidation of lipids present on cell membranes, resulting in cell damage. Lipid peroxidation causes formation of reactive aldehydes including MDA, which can be used to assess oxidative stress. The MDA assay was carried out using a commercially available kit (ab118970, Abcam) following the manufacturer's instructions. Briefly, 200 μl of cell culture supernatant was aliquoted for the assay. MDA standards were also prepared. Then 600 μl of the thiobarbituric acid reagent was added both to the samples and to the MDA standards and incubated at 95°C for 60 min. Tubes were cooled to room temperature using an ice bath for 10 min, 200 μl of the cooled solution was added to one well of 96-well plates (three biological replicates), and the absorbance was immediately measured on a microplate reader to calculate the OD at 532 nm. The standard curve was plotted, and the MDA concentrations of the samples were calculated. Increased MDA concentration is associated with increased oxidative stress.

The SOD assay was carried out using the commercially available SOD kit (Abcam, Waltham, MA, USA) following the manufacturer's instructions. The cell culture medium was collected and 20 μl of each sample was used for analysis. Samples were prepared with and without addition of SOD enzyme solution for the blank correction. To the 20 μl sample, 200 μl of the WST working solution was added. The assay uses a tetrazolium salt WST-1, which produces a water-soluble formazan dye upon reduction with superoxide anion. The WST-1 reduction is linearly related to SOD-mediated inhibition activity of xanthine oxidase. SOD catalyzes the dismutation of the superoxide anion into hydrogen peroxide and molecular oxygen, resulting in decrease of WST-1 reduction. The inhibition activity of SOD was measured by colorimetric measurement of OD at 450 nm.

• Schematic Diagram

Supplier Data

Schematic Diagram - Superoxide Dismutase Activity Assay Kit (Colorimetric) (AB65354)

Representative image of Superoxide Dismutase Activity Assay Kit (Colorimetric) ab65354

Components shown from left to right :

- SOD Enzyme Solution

- WST Reagent II

- SOD Dilution Buffer

- SOD Assay Buffer

Note : The vial labels shown in this image use generic names for illustrative purposes only and may not exactly match the specific component names included in the kit.

Note : Colors of solutions in image may not precisely match the shade of colors in the actual kit.