Amplite® Colorimetric Superoxide Dismutase (SOD) Assay Kit

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.

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.

Citations

View all 8 citations: Citation Explorer

Intracellular pyruvate as one of the major bioactive substances of lactic acid bacteria isolated from kimchi
Authors: Kang, Jin Yong and Lee, Moeun and Song, Jung Hee and Choi, Eun Ji and Mun, So Yeong and Kim, Daun and Lim, Seul Ki and Kim, Namhee and Park, Bo Yeon and Chang, Ji Yoon
Journal: Journal of Food Science (2024)

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

References

View all 111 references: Citation Explorer

Cu,Zn superoxide dismutase and zinc stress in the metal-tolerant ericoid mycorrhizal fungus Oidiodendron maius Zn
Authors: Vallino M, Martino E, Boella F, Murat C, Chiapello M, Perotto S.
Journal: FEMS Microbiol Lett (2009): 48

In vivo, in vitro, and in silico studies of Cu/Zn-superoxide dismutase regulation by molecules in grape seed procyanidin extract
Authors: Puiggros F, Sala E, Vaque M, Ardevol A, Blay M, Fern and ez-Larrea J, Arola L, Blade C, Pujadas G, Salvado MJ.
Journal: J Agric Food Chem (2009): 3934

Functional polymorphism in manganese superoxide dismutase and antioxidant status: their interactions on the risk of cervical intraepithelial neoplasia and cervical cancer
Authors: Tong SY, Lee JM, Song ES, Lee KB, Kim MK, Lee JK, Son SK, Lee JP, Kim JH, Kwon YI.
Journal: Gynecol Oncol (2009): 272

Imidazolate-bridged dicopper(II) and copper(II)-zinc(II) complexes of macrocyclic ligand with methylimidazol pendants: Model study of copper(II)-zinc(II) superoxide dismutase
Authors: Yuan Q, Cai K, Qi ZP, Bai ZS, Su Z, Sun WY.
Journal: J Inorg Biochem (2009): 1156

Characterization, molecular modelling and developmental expression of zebrafish manganese superoxide dismutase
Authors: Lin CT, Tseng WC, Hsiao NW, Chang HH, Ken CF.
Journal: Fish Shellfish Immunol (2009): 318

Page updated on November 21, 2024

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