Amplite® Colorimetric Superoxide Dismutase (SOD) Assay Kit

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Alternative formats

Amplite® Colorimetric Superoxide Dismutase (SOD) Assay Kit *Enhanced Sensitivity*

Overview SDS Protocol

Superoxide dismutases (SOD) are a class of enzymes that catalyze the dismutation of superoxide into oxygen and hydrogen peroxide. Superoxide is one of the main reactive oxygen species in cells. It is a substantial contributor of pathology associated with neurodegenerative diseases, ischemia reperfusion injury, atherosclerosis and aging. SODs are an important antioxidant defense in nearly all cells exposed to superoxide radicals. In fact, mice lacking SOD1 develop a wide range of pathologies, including hepatocellular carcinoma, an acceleration of age-related muscle mass loss, an earlier incidence of cataracts and a reduced lifespan. Overexpression of SOD protects murine fibrosarcoma cells from apoptosis and promotes cell differentiation. The Amplite® Colorimetric Superoxide Dismutase (SOD) Assay Kit provides a quick and sensitive method for the measurement of SOD activity in solutions. In the assay, xanthine is converted to superoxide radical ions, uric acid and hydrogen peroxide by xanthine oxidase (XO). Superoxide reacts with ReadiView™ SOD560 to generate a product that absorbs around 560 nm. SOD inhibits the reaction of ReadiView™ SOD560 with superoxide, thus reduces the absorption at 560 nm. The reduction in the absorption of ReadiView™ SOD560 at 560 nm is proportional to SOD activity. The kit can be performed in a convenient 96-well or 384-well microtiter-plate format.

Platform

Absorbance microplate reader

Absorbance	560 nm
Recommended plate	Clear bottom

Components

Example protocol

AT A GLANCE

Protocol Summary

Prepare SOD standards or test samples (50 µL)
Add SOD working solution 1 (25 µL)
Add SOD working solution 2 (25 µL)
Incubate at room temperature for 30 - 60 minutes
Monitor absorbance at 560 nm

Important Thaw all the kit components at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

SOD standard solution (10 kU/mL)

Add 50 µL of Assay Buffer (Component E) into the vial of SOD Standard (Component D) to make 10 kU/mL standard solution.

PREPARATION OF STANDARD SOLUTION

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/11305

SOD standard

Add 10 µL of 10 kU/mL SOD standard solution into 990 µL of Assay Buffer (Component E) to get 100 U/mL SOD standard solution (SD7). Take 100 U/mL SOD standard solution (SD7) and perform 1:10 in Assay Buffer (Component E) to get 10 U/mL SOD standard solution (SD6). Take 10 U/mL standard solution (SD6) and perform 1:3 serial dilutions to get serially diluted SOD standards (SD5 - SD1) with Assay Buffer (Component E).

PREPARATION OF WORKING SOLUTION

1. SOD working solution 1

Add 2.5 mL of Assay Buffer (Component E) into the bottle of ReadiView^TM SOD560 (Component A) and mix well. Then add 50 μL of 50X Xanthine (Component B) into this bottle to make SOD working solution 1.
Note This SOD working solution 1 should be prepared before the experiment, and kept from light. SOD working solution 1 is not stable and the unused portion should be discarded.

2. SOD working solution 2

Add 50 μL Assay Buffer (Component E) into the vial of Xanthine Oxidase (Component C) and mix well. Then, transfer 50 μL of Xanthine Oxidase stock solution into 2.5 mL Assay Buffer (Component E) to make SOD working solution 2.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of SOD standards and test samples in a clear bottom 96-well microplate. SD=SOD Standards (SD1 - SD7, 0.041 to 100 U/mL); BL=Blank Control; TS=Test Samples.

BL	BL	TS	TS
SD1	SD1	...	...
SD2	SD2	...	...
SD3	SD3
SD4	SD4
SD5	SD5
SD6	SD6
SD7	SD7

Table 2. Reagent composition for each well.

Well	Volume	Reagent
SD1 - SD7	50 µL	Serial Dilution (0.041 to 100 U/mL)
BL	50 µL	Assay Buffer (Component E)
TS	50 µL	test sample

Prepare SOD standards (SD), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
Add 25 µL of SOD working solution 1 to each well of SOD standard, blank control, and test samples to make the total assay volume of 75 µL/well. For a 384-well plate, add 12.5 µL of SOD working solution 1 into each well instead, for a total volume of 37.5 µL/well.
Add 25 µL of SOD working solution 2 to each well of SOD standard, blank control, and test samples to make the total assay volume of 100 µL/well. For a 384-well plate, add 12.5 µL of SOD working solution 2 into each well instead, for a total volume of 50 µL/well.
Incubate the reaction at room temperature for 30 to 60 minutes, protected from light.
Monitor the absorbance with an absorbance plate reader at 550 to 560 nm.

Images

Figure 1. SOD dose response was measured with Amplite® Colorimetric Superoxide Dismutase Assay Kit in a 96-well white wall/clear bottom plate with a Spectrum Max microplate reader (Molecular Devices).

Figure 2. SOD dose response was measured with Amplite® Colorimetric Superoxide Dismutase Assay Kit in a 96-well white wall/clear bottom plate with a Spectrum Max microplate reader (Molecular Devices). As low as 0.1 U/mL SOD was detected with 60 minutes incubation time (n=3). The figure is SOD activity as a function of absorbance at 560 nm (OD).

Figure 3. Effect of gallic acid on oxidative stress. Liver endogenous antioxidant levels of SOD. The SOD enzyme activity was estimated using Amplite Colorimetric Superoxide Dismutase Assay Kit (Cat no 11305, AAT Bioquest, Inc., USA). Data are shown as mean ± SEM (n = 6/group). Statistical analysis was performed using one-way ANOVA followed by Tukey-Kramer multiple comparisons test. #Significantly different from control group (P < 0.001), *Significantly different from toxin group (p < 0.05), **Significantly different from toxin group (p < 0.01), ***Significantly different from toxin group (p < 0.001). Toxin group- isoniazid and rifampicin group, GA 50 + T- Gallic acid (50 mg/kg) + toxin, GA 100 + T- Gallic acid (100 mg/kg) + toxin, GA 150 + T- Gallic acid (150 mg/kg) + toxin, Sil + T- Silymarin (100 mg/kg) + toxin. Source: Gallic acid attenuates isoniazid and rifampicin-induced liver injury by improving hepatic redox homeostasis through influence on Nrf2 and NF-κB signalling cascades in Wistar Rats by Sukumaran Sanjay, Chandrashekaran Girish, Pampa Ch Toi, Zachariah Bobby. Journal of Pharmacy and Pharmacology, April 2021

Citations

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Astragaloside IV Reduces Mutant Ataxin-3 Levels and Supports Mitochondrial Function in Spinocerebellar Ataxia Type 3
Authors: Lin, Yongshiou and Cheng, Wenling and Chang, Juichih and Wu, Yuling and Hsieh, Mingli and Liu, Chin-San
Journal: (2023)

Amelioration of AlCl3-induced Memory Loss in the Rats by an Aqueous Extract of Guduchi, a Medhya Rasayana
Authors: Jamadagni, Shrirang B and Ghadge, Pooja M and Tambe, Mukul S and Srinivasan, Marimuthu and Prasad, Goli Penchala and Jamadagni, Pallavi S and Prasad, Shyam Baboo and Pawar, Sharad D and Gurav, Arun M and Gaidhani, Sudesh N and others,
Journal: Pharmacognosy Magazine (2023): 09731296221145063

Comparison of the Probiotic Potential between Lactiplantibacillus plantarum Isolated from Kimchi and Standard Probiotic Strains Isolated from Different Sources
Authors: Jeong, Chang-Hee and Sohn, Hyejin and Hwang, Hyelyeon and Lee, Ho-Jae and Kim, Tae-Woon and Kim, Dong-Sub and Kim, Chun-Sung and Han, Sung-Gu and Hong, Sung-Wook
Journal: Foods (2021): 2125

Propagation of Mitochondria-Derived Reactive Oxygen Species within the Dipodascus magusii Cells
Authors: Rogov, Anton G and Goleva, Tatiana N and Epremyan, Khoren K and Kireev, Igor I and Zvyagilskaya, Renata A
Journal: Antioxidants (2021): 120

Gallic acid attenuates isoniazid and rifampicin-induced liver injury by improving hepatic redox homeostasis through influence on Nrf2 and NF-$\kappa$B signalling cascades in Wistar Rats
Authors: Sanjay, Sukumaran and Girish, Chandrashekaran and Toi, Pampa Ch and Bobby, Zachariah
Journal: Journal of Pharmacy and Pharmacology (2021): 473--486

Propagation of Mitochondria-Derived Reactive Oxygen Species within the Dipodascus magnusii Cells
Authors: Rogov, Anton G and Goleva, Tatiana N and Epremyan, Khoren K and Kireev, Igor I and Zvyagilskaya, Renata A
Journal: Antioxidants (2021): 120

Intercomparative investigation of the total phenols, total flavonoids, In vitro and In vivo antioxidant activities of capparis Cartilaginea (Decne.) maire and weiller and Capparis Ovata Desf. from Jordan
Authors: Al-Qudah, Mahmoud A and Obeidat, Safwan M and Muhaidat, Riyadh and Al-Trad, Bahaa and Al-Jaber, Hala I and Lahham, Jamil N and others, undefined
Journal: Pharmacognosy Magazine (2018): 154

References

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Functional polymorphism in manganese superoxide dismutase and antioxidant status: their interactions on the risk of cervical intraepithelial neoplasia and cervical cancer
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Synthesis, characterization and potent superoxide dismutase-like activity of novel bis(pyrazole)-2,2'-bipyridyl mixed ligand copper(II) complexes
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Effects of Chinese herbal medicine combined with He-Ne laser on lipoperoxide and superoxide dismutase in chloasma patients
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The p.E22G mutation in the Cu/Zn superoxide-dismutase gene predicts a long survival time: clinical and genetic characterization of a seven-generation ALS1 Spanish pedigree
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Association between manganese superoxide dismutase gene polymorphism and risk of prostate cancer: a meta-analysis
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