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
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
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 ReadiViewTM 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.
Table 2. Reagent composition for each well.
BL | BL | TS | TS |
SD1 | SD1 | ... | ... |
SD2 | SD2 | ... | ... |
SD3 | SD3 | ||
SD4 | SD4 | ||
SD5 | SD5 | ||
SD6 | SD6 | ||
SD7 | SD7 |
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 9 citations: Citation Explorer
Erinacine A-Enriched Hericium erinaceus Mycelium Ethanol Extract Lessens Cellular Damage in Cell and Drosophila Models of Spinocerebellar Ataxia Type 3 by Improvement of Nrf2 Activation
Authors: Wu, Yu-Ling and Sun, Hai-Lun and Chang, Jui-Chih and Lin, Wei-Yong and Chen, Pei-Yin and Chen, Chin-Chu and Lee, Li-Ya and Li, Chien-Chun and Hsieh, Mingli and Chen, Haw-Wen and others,
Journal: Antioxidants (2024): 1495
Authors: Wu, Yu-Ling and Sun, Hai-Lun and Chang, Jui-Chih and Lin, Wei-Yong and Chen, Pei-Yin and Chen, Chin-Chu and Lee, Li-Ya and Li, Chien-Chun and Hsieh, Mingli and Chen, Haw-Wen and others,
Journal: Antioxidants (2024): 1495
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)
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)
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
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
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
References
View all 111 references: Citation Explorer
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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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
Molecular characterization of a cDNA encoding Cu/Zn superoxide dismutase from Deschampsia antarctica and its expression regulated by cold and UV stresses
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Journal: BMC Res Notes (2009): 198
Authors: Sanchez-Venegas JR, Dinamarca J, Moraga AG, Gidekel M.
Journal: BMC Res Notes (2009): 198
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
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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
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Journal: Fish Shellfish Immunol (2009): 318
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Journal: Fish Shellfish Immunol (2009): 318
Superoxide dismutase activity of corrole metal complexes
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Journal: Dalton Trans (2009): 7879
Authors: Eckshtain M, Zilbermann I, Mahammed A, Saltsman I, Okun Z, Maimon E, Cohen H, Meyerstein D, Gross Z.
Journal: Dalton Trans (2009): 7879
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