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Frequently Asked Questions for BIOXYTECH®
Superoxide Dismutase
Catalog Number: 21010
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How long does the test take to perform?
15 minutes for sample preparation (10 min. of this involves centrifugation), 2 minutes to add reagents and read. Total procedure time is <20 minutes.

What is the assay range
0.1 - 13 SOD-525 units per mL assay volume.

What samples can be tested for SOD?
Any sample can be tested as long as the correct sample preparation steps are taken as described in the sample preparation protocol found in the package insert. However, the most common samples are erythrocyte lysates or homogenates of various animal tissues. When assaying cells other than erythrocytes 2.0 x 107 is a useful starting point however SOD concentration will vary depending on the model system. Therefore it is up to the researcher to determine appropriate cell sample range.

How are samples prepared prior to testing?
Excess amounts of hemoglobin, albumin, ascorbic acid, NADPH, and BHT that may interfere with the assay are removed from the sample by a well defined protocol. See package insert.

Can the SOD-525 assay be used to discriminate between the different forms on superoxide dismutase?
The assay does not discriminate between Cu/Zn, Mn, or Fe forms, however it does become specific for Cu/Zn form if ethanol/chloroform extraction is performed. If the user wishes to assay for Mn SOD then the appropriate cell fraction would need to be isolated (i.e. mitochondria). The extraction step, while necessary to remove interference due to hemoglobin, introduces a 1.475 added dilution factor. The ethanol in the extracted sample also decreases the measured absorbance requiring that sample blanks and controls be extracted as well.

What concentrations of SOD can be found in various samples?
SOD can be found at various concentrations in the following samples: 80 - 100 U-525/ml of human erythrocyte lysate 115 - 135 U-525/ml rat erythrocyte lysate ~73 U-525/ml of rat liver extract per mg protein ~35 U-525/ml of rat heart extract per mg protein ~50 U-525/ml of rat kidney extract per mg of protein All values based on testing of "normal" samples.

What is the stability of the assay reaction?
The SOD-525 assay requires the measurement in absorbance change per unit time. After a 1 minute incubation the absorbance must be measured every six seconds out to 1 minute. If the sample is too concentrated there may be significant lag time wherein no absorbance change is noted. In this case dilute the sample and re-test.

What control is needed to perform the test?
A sample of water (no SOD activity) is used as a control for the rate of the test reaction. The control rate is compared to the rate of samples containing enzyme activity. For those wanting a standard, OXIS supplies Cu/Zn SOD Catalog No: 27619

What is the shelf life of the SOD-525 kit?
1 year after the date of manufacture. 6 weeks after vials are opened.

How is SOD calculated in the procedure?
There are 2 choices available to calculate SOD activity: The absorbance reading of the sample (i.e., kinetic rate of Vs) is divided by the absorbance reading of the control (i.e., kinetic rate of Vc). A value corresponding to this ratio can be found on the chart in the package insert. Next to this value is a corresponding unit of SOD activity. An alternative method for calculating SOD activity can be derived from the following rate equation: Vs/Vc = 1+ ([SOD] / a [SOD] + b) with a = 0.073 1 + b = 0.93 The concentration for SOD is solved as follows: [SOD] = [0.93 (Vs/Vc-1)] / [1.073 - 0.073(Vs/Vc)]

How does the SOD-525 kit compare to other methods?
The ferri-cytochrome c/xanthine oxidase assay is considered the reference method. It has the following disadvantages: Commercial cytochrome c is contaminated with SOD. Tissue extracts contain quinone which interfere with the assay. It takes 45 minutes to 90 minutes to normalize the blank. The rate measurement of each sample takes 4 to 5 minutes. Other commercially available kits: Similar sensitivity as the SOD-525 assay, but more time consuming and labor intensive, requiring the preparation of a standard curve.

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