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

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. Amplite® Colorimetric Superoxide Dismutase (SOD) Assay Kit provides a rapid and sensitive method for the measurement of SOD activity. It is well-known that NADH and SOD enzyme system generates superoxide radicals that reduce WST-1 into a yellow color formazan dye that has maximum absorption around 440 nm. SOD inhibits the reduction of WST-1 by catalyzing the dismutation of the superoxide anion into hydrogen peroxide and molecular oxygen, thus reduces the 440 nm absorption of the formazan product. The reduction of 440 nm absorption 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 and add 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 440 nm

Important Thaw one of each kit component 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/11308

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 10U/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 WST-1 (Component A) and mix well. Then add 50 μL of 50X SOD Enzyme solution (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 NADH (Component C) and mix well. Then, transfer 50 μL of NADH 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 (SD7 - SD1, 100 to 0.041 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 (100 to 0.041 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 440 nm.

Citations

View all 33 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)

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

Accelerated sarcopenia in Cu/Zn superoxide dismutase knockout mice
Authors: Deepa, S. S., Van Remmen, H., Brooks, S. V., Faulkner, J. A., Larkin, L., McArdle, A., Jackson, M. J., Vasilaki, A., Richardson, A.
Journal: Free Radic Biol Med (2019): 19-23

The copper-zinc superoxide dismutase activity in selected diseases
Authors: Lew, undefined and owski, L., Kepinska, M., Milnerowicz, H.
Journal: Eur J Clin Invest (2019): e13036

Carcinogenesis and Reactive Oxygen Species Signaling: Interaction of the NADPH Oxidase NOX1-5 and Superoxide Dismutase 1-3 Signal Transduction Pathways
Authors: Parasc, undefined and olo, A., Laukkanen, M. O.
Journal: Antioxid Redox Signal (2019): 443-486

References

View all 111 references: Citation Explorer

Association of cigarette smoking with superoxide dismutase enzyme levels in subjects with chronic periodontitis
Authors: Agnihotri R, P and urang P, Kamath SU, Goyal R, Ballal S, Shanbhogue AY, Kamath U, Bhat GS, Bhat KM.
Journal: J Periodontol (2009): 657

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

Enhancement of aglycone, vitamin K2 and superoxide dismutase activity of black soybean through fermentation with Bacillus subtilis BCRC 14715 at different temperatures
Authors: Wu CH, Chou CC.
Journal: J Agric Food Chem (2009): 10695

High-level molecular diversity of copper-zinc superoxide dismutase genes among and within species of arbuscular Mycorrhizal fungi
Authors: Corradi N, Ruffner B, Croll D, Colard A, Horak A, S and ers IR., undefined
Journal: Appl Environ Microbiol (2009): 1970

Superoxide dismutase and catalase activity in naturally derived commercial surfactants
Authors: Dani C, Buonocore G, Longini M, Felici C, Rodriguez A, Corsini I, Rubaltelli FF.
Journal: Pediatr Pulmonol (2009): 1125

Page updated on November 21, 2024

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