Superoxide Dismutase (SOD) Colorimetric Activity Kit

Catalog No. ABIN2866576

Product Details

Target: SOD1 (Superoxide Dismutase 1, Soluble (SOD1))

Reactivity: Human, Various Species

Detection Method: Colorimetric

Application: Activity Assay (AcA)

Purpose: The DetectX® Superoxide Dismutase (SOD) Activity Kit is designed to quantitatively measure SOD activity in a variety of samples.

Brand: DetectX®

Sample Type: Serum, Plasma, Cell Samples, Tissue Samples, Cell Lysate

Components: Clear 96 well Half Area Plates 2 Plates
Superoxide Dismutase Standard 1 vial 1 Unit/vial of bovine Erythrocyte Superoxide Dismutase lyophilized.
Assay Buffer 50 mL Buffer containing detergents, stabilizers and dye.
Xanthine Oxidase Buffer 6 mL Buffer containing detergents and stabilizers.
Xanthine Oxidase Concentrate 225 μL A 25X concentrated suspension of Xanthine Oxidase.
Substrate Diluent 12 mL Substrate buffer.
Substrate Concentrate 1.1 mL .1ML A 10X concentrate of the Detection Reagent.

Material not included: 2 mM Potassium Cyanide solution for inhibition of Cu/Zn and extracellular SOD if desired.
Repeater pipet with disposable tips capable of dispensing 25 and 50 μL. 96 well plate reader capable of reading optical absorption at 450 nm.
Software for converting optical density (OD) readings from the plate reader and carrying out four parameter logistic curve (4PLC) fitting.

Application Details

Protocol: The assay measures all types of SOD activity, including Cu/Zn, Mn, and FeSOD types.
Please read the complete kit insert before performing this assay.
A bovine erythrocyte SOD standard is provided to generate a standard curve for the assay and all samples should be read off of the standard curve.
Samples are diluted in our specially colored Sample Diluent and added to the wells.
The Substrate is added followed by Xanthine Oxidase Reagent and incubated at room temperature for 20 minutes.
The Xanthine Oxidase generates superoxide in the presence of oxygen, which converts a colorless substrate in the Detection Reagent into a yellow colored product.
The colored product is read at 450 nm.
Increasing levels of SOD in the samples causes a decrease in superoxide concentration and a reduction in yellow product.
The activity of the SOD in the sample is calculated after making a suitable correction for any dilution, using software available with most plate readers.
The results are expressed in terms of units of SOD activity per mL.

Sample Preparation:

Samples should be kept on ice to maintain enzyme activity. Cell Suspensions and Adherent Cells 1. Centrifuge > 1 x 106 cells in suspension at 250 x g for 10 minutes at 4 °C. Discard the supernatant. 2. Resuspend the cell pellet in ice-cold PBS and transfer to a microtube on ice. Centrifuge, discard supernatant, and place on ice. 3. Wash > 1 x 106 adherent cells with PBS prior to being harvested by gentle trypsinization. Transfer to a tube on ice and centrifuge at 250 x g for 10 minutes at 4 °C and discard the supernatant. 4. Wash the pellet with ice-cold PBS and centrifuge at 250 x g for 10 minutes at 4 °C. 5. Homogenize or sonicate the pellet in 0.5-1 mL of PBS per 100 mg of cells. Centrifuge at 1,500 x g for 10 minutes at 4 °C. 6. Collect the supernatant and assay immediately, or store at -80 °C. Dilute at least 1:4 in Assay Buffer prior to measuring SOD activity. A 1:4 dilution of the sample is made by adding 3 parts of Assay Buffer to 1 part of supernatant.

Assay Procedure:

Use the plate layout sheet on the back page to aid in proper sample and standard identification. The Assay Buffer contains detergents. When pipetting samples or standards into the wells carefully add the sample slowly down the side of the well. Use Reverse Pipetting to avoid bubbles!
1. Pipet 10 μL of samples or appropriate standards into duplicate wells in the plate.
2. Pipet 10 μL of Assay Buffer into duplicate wells as the Zero standard.
3. Add 50 μL of the Substrate Preparation to each well using a repeater pipet. NOTE: If your samples have significant yellow coloration then pre-read the optical density at 450 nm.
4. Add 25 μL of the Xanthine Oxidase Preparation to each well using a repeater pipet.
5. Incubate at room temperature for 20 minutes.
6. Read the optical density at 450 nm.
Reverse pipetting involves pressing the plunger down to the blow-out prior to picking up stan- dards and samples. The selected volume of liquid plus an excess is pulled into the pipette tip. To dispense, the plunger is pressed only down to the first position, leaving some liquid in the tip. This way, liquid remains inside the tip when dispensing, minimizing bubble formation.

Calculation of Results:

Average the duplicate OD readings for each standard and sample.
Or use online tools
If your sample was visibly colored, then subtract the pre-read optical density at 450 nm from the subsequent Xanthine Oxidase reaction optical density after 20 minutes.
Inhibition values are sometimes quoted for SOD activity.
Inhibition values can be obtained by dividing the measured Mean OD for the standard or samples by the Mean OD for the Zero standard (No SOD) and multiplying the result by 100.
Create a standard curve by reducing the data using computer software capable of generating a four-parameter logistic curve (4PLC) fit.
The sample activities obtained should be multiplied by the dilution factor to obtain neat sample values. typical data Sample Mean OD % Inhibition SOD Activity (U/mL) Standard 1 0.075 10.5 4 Standard 2 0.110 15.4 2 Standard 3 0.169 23.7 1 Standard 4 0.262 36.7 0.5 Standard 5 0.359 50.4 0.25 Standard 6 0.480 67.3 0.125 Standard 7 0.568 79.7 0.0625 Zero 0.713 100 0 Sample 1 0.231 32.4 0.605 Sample 2 0.168 23.6 1.030 Always run your own standard curves for calculation of results.
Do not use these data.
SOD Unit Definition One unit of SOD is defined as the amount of enzyme causing half the maximum inhibition of the reduction of 1.5 mM Nitro blue tetrazolium in the presence of riboflavin at 25 °C and pH 7.8.

Restrictions: For Research Use only

Handling

Precaution of Use: As with all such products, this kit should only be used by qualified personnel who have had labo- ratory safety instruction.
The complete insert should be read and understood before attempting to use the product.
Sample typeS and preparatiOn Samples that need to be stored after collection should be stored at -70°C or lower, preferably after being frozen in liquid nitrogen.
This assay has been validated for serum, plasma and erythrocyte lysates.
Samples containing visible particulate should be centrifuged prior to using.
Some serum and plasma samples may contain significant hemoglobin concentrations and the optical density at 450nm determined prior to running the assay.
After addition of the Substrate solution to all the used wells the optical density at 450 nm should be read and subtracted from the optical density recorded at the end of the 20 minute incubation.
Process any cell pellet as described for Cell Lysates on page 7.
To measure cytosolic (SOD1, Cu/Zn) and/or mitochondrial SOD (SOD2, Mn) the sample superna- tants prepared on page 7 should be centrifuged at 10,000 x g for 15 minutes at 4°C.
The superna- tants will contain the cytosolic SOD and the cell pellets will contain mitochondrial SOD.
To deter- mine Mn SOD (SOD2) activity treat samples with 2 mM potassium cyanide.
Addition of cyanide will inactivate other SOD enzymes.
Iron containing SODs (FeSOD) are found in some bacteria and plants and have similar properties to MnSOD (SOD2).
Extracellular SOD (SOD3) is obtained from serum, plasma, ascites or synovial fluid fluids.

Storage: -20 °C,4 °C

Storage Comment: All components of this kit should be stored at 4°C until the expiration date of the kit. Once reconstituted, the Superoxide Dismutase Standard should be aliquoted and stored at -20°C.

References

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Target Details

Target: SOD1 (Superoxide Dismutase 1, Soluble (SOD1))

Alternative Name: Superoxide Dismutase (SOD) (SOD1 Products)

Synonyms: LOC692639 Kit, ALS Kit, ALS1 Kit, IPOA Kit, SOD Kit, hSod1 Kit, homodimer Kit, CG11793 Kit, Cu Kit, Cu-Zn SOD Kit, Cu/Zn SOD Kit, Cu/Zn sod Kit, Cu/Zn superoxide dismutase Kit, Cu/ZnSOD Kit, CuSOD Kit, CuZn SOD Kit, CuZn-SOD Kit, CuZn-SOD1 Kit, CuZnSOD Kit, Cu[2+]/Zn[2+]SOD Kit, Dmel\\CG11793 Kit, G Kit, Mn SOD Kit, SOD-1 Kit, SOD1 Kit, Sod-1 Kit, Sod1 Kit, To Kit, To-1 Kit, Zn SOD Kit, Zn Sod Kit, Zn-SOD Kit, ZnSod Kit, cSOD Kit, cSod Kit, dSOD1 Kit, l(3)108 Kit, l(3)68Af' Kit, l(3)G Kit, sod Kit, sod1 Kit, CU/ZN-SOD Kit, SODC Kit, DKFZP469M1833 Kit, B430204E11Rik Kit, Cu/Zn-SOD Kit, Ipo-1 Kit, Ipo1 Kit, SOD1L1 Kit, XSODB Kit, als Kit, als1 Kit, ipoa Kit, sod1-a Kit, ZSOD Kit, cuzn Kit, Cu/Zn superoxide dismutase Kit, superoxide dismutase 1 Kit, Superoxide dismutase 1 Kit, superoxide dismutase 1, soluble Kit, superoxide dismutase 1 S homeolog Kit, Superoxide dismutase [Cu-Zn] Kit, superoxide dismutase 1 L homeolog Kit, superoxide dismutase [Cu-Zn]-like Kit, superoxide dismutase [Cu-Zn] Kit, superoxide dismutase Sod1 Kit, SOD Kit, SOD1 Kit, Sod1 Kit, sod1 Kit, sod1.S Kit, sod-1 Kit, sod1.L Kit, LOC101451855 Kit, LOC101115136 Kit

Background: Short-lived and highly reactive oxygen species Superoxide ion (ROS) such as O •- (superoxide), •OH (hydroxyl .2 2O + 2e- - - +radical), and H 2 O2 + O2 2H2O2 (hydrogen peroxide) are continuously generated in vivo. In the resting state, NADH:Ubiquinone Oxidoreductase the balance between antioxidants and oxidants or Ubiquinol:Cyt c is sufficient to prevent the disruption of normal Reductase SOD physiologic functions, however, either increases in oxidants or decreases in antioxidants can Oxidation/Reduction disrupt this balance giving rise to elevated levels Mechanisms O + H O of reactive oxygen species (ROS)1,2. 2 2 2 The cellular levels of ROS are controlled by antioxidant enzymes and small molecule antioxidants. The major antioxidant enzymes, superoxide dismutases (SODs), including copper-zinc superoxide dismutase (Cu/ZnSOD, SOD1), manganese superoxide dismutase (MnSOD, SOD2) and extracel- lular superoxide dismutase (EC-SOD, SOD3), all play critical roles in scavenging O -2• . Decreased SOD activity results in elevated level of superoxide which in turn leads to decreased NO but in- creased peroxynitrite concentrations. The major intracellular SOD is a 32-kD copper and zinc con- taining homodimer (Cu/Zn SOD). The mitochondrial SOD (MnSOD) is a manganese-containing 93-kD homotetramer that is synthesized in the cytoplasm and translocated to the inner matrix of mitochondria. EC-SOD is the primary extracellular SOD enzyme and is highly expressed in many organs. Increased SOD activity levels are seen in Downs Syndrome3 while decreased activity is seen in diabetes, Alzheimer's disease, rheumatoid arthritis, Parkinson's disease, uremic anemia, atherosclerosis, some cancers, and thyroid dysfunction3-8