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

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Amplite® Colorimetric Superoxide Dismutase (SOD) Assay Kit

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. 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.

Platform

Absorbance microplate reader

Absorbance	440 nm
Recommended plate	Clear bottom

Components

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.

Images

Figure 1. OD 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).

Citations

View all 32 citations: Citation Explorer

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

Evaluation and Monitoring of Superoxide Dismutase (SOD) Activity and its Clinical Significance in Gastric Cancer: A Systematic Review and Meta-Analysis
Authors: Li, J., Lei, J., He, L., Fan, X., Yi, F., Zhang, W.
Journal: Med Sci Monit (2019): 2032-2042

Utilizing Superoxide Dismutase Mimetics to Enhance Radiation Therapy Response While Protecting Normal Tissues
Authors: Mapuskar, K. A., Anderson, C. M., Spitz, D. R., Batinic-Haberle, I., Allen, B. G., E. Oberley-Deegan R
Journal: Semin Radiat Oncol (2019): 72-80

Superoxide dismutase: a review and a modified protocol for activities measurements in rat livers
Authors: Campos-Shimada, L. B., Hideo Gilglioni, E., Fern and es Garcia, R., Rizato Martins-Maciel, E., Luiza Ishii-Iwamoto, E., Luzia Salgueiro-Pagadigorria, C.
Journal: Arch Physiol Biochem (2018): 1-8

Human Mn-superoxide dismutase inactivation by peroxynitrite: a paradigm of metal-catalyzed tyrosine nitration in vitro and in vivo
Authors: Demicheli, V., Moreno, D. M., Radi, R.
Journal: Metallomics (2018): 679-695

A Review of the Catalytic Mechanism of Human Manganese Superoxide Dismutase
Authors: Azadmanesh, J., Borgstahl, G. E. O.
Journal: Antioxidants (Basel) (2018): se name="11308.enl" path="C:\Users\aatbi\Drop

Inhibition of copper-zinc superoxide dismutase activity by selected environmental xenobiotics
Authors: Lew, undefined and owski, L., Kepinska, M., Milnerowicz, H.
Journal: Environ Toxicol Pharmacol (2018): 105-113

References

View all 111 references: Citation Explorer

Technical note: copper chaperone for copper, zinc superoxide dismutase: a potential biomarker for copper status in cattle
Authors: Hepburn JJ, Arthington JD, Hansen SL, Spears JW, Knutson MD.
Journal: J Anim Sci (2009): 4161

Tissue expression of manganese superoxide dismutase is a candidate prognostic marker for glioblastoma
Authors: Park CK, Jung JH, Moon MJ, Kim YY, Kim JH, Park SH, Kim CY, Paek SH, Kim DG, Jung HW, Cho BK.
Journal: Oncology (2009): 178

Expression of human Cu, Zn-superoxide dismutase in an insect cell-free system and its structural analysis by MALDI-TOF MS
Authors: Ezure T, Suzuki T, Ando E, Nishimura O, Tsunasawa S.
Journal: J Biotechnol (2009): 287

Normal-tissue radioprotection by overexpression of the copper-zinc and manganese superoxide dismutase genes
Authors: Veldwijk MR, Herskind C, Sellner L, Radujkovic A, Laufs S, Fruehauf S, Zeller WJ, Wenz F.
Journal: Strahlenther Onkol (2009): 517

Regulation of Mn-superoxide dismutase activity and neuroprotection by STAT3 in mice after cerebral ischemia
Authors: Jung JE, Kim GS, Narasimhan P, Song YS, Chan PH.
Journal: J Neurosci (2009): 7003

High levels of autoantibodies against catalase and superoxide dismutase in nasopharyngeal carcinoma
Authors: Gargouri B, Lassoued S, Ben Mansour R, Ayadi W, Idriss N, Attia H, El Feki Ael F.
Journal: South Med J (2009): 1222

Lecithinized superoxide dismutase suppresses free radical substrates during the early phase of burn care in rats
Authors: Koizumi T, Goto H, Tanaka H, Yamaguchi Y, Shimazaki S.
Journal: J Burn Care Res (2009): 321

Different extent of cardiac malfunction and resistance to oxidative stress in heterozygous and homozygous manganese-dependent superoxide dismutase-mutant mice
Authors: Loch T, Vakhrusheva O, Piotrowska I, Ziolkowski W, Ebelt H, Braun T, Bober E.
Journal: Cardiovasc Res (2009): 448

Metallopeptide based mimics with substituted histidines approximate a key hydrogen bonding network in the metalloenzyme nickel superoxide dismutase
Authors: Shearer J, Neupane KP, Callan PE.
Journal: Inorg Chem (2009): 10560

Superoxide dismutase is regulated by LAMMER kinase in Drosophila and human cells
Authors: James BP, Staatz WD, Wilkinson ST, Meuillet E, Powis G.
Journal: Free Radic Biol Med (2009): 821