Superoxide Dismutase (SOD) ELISA Kit

Antigen: Superoxide Dismutase (SOD)

Synonyms: CG8905, DmelCG8905, dSOD2, Mn SOD, Mn-SOD, Mn-SOD2, mnSOD, MnSOD, MnSODII, Mn[2+]SOD, SOD, Sod-2, SOD-2, SOD2, Cu, Zn Sod, Zn-SOD, Cu-Zn SOD, Cu/Zn SOD, Cu/Zn superoxide dismutase, Cu/ZnSOD, CuSOD, CuZn SOD, CuZn-SOD, CuZnSOD, Cu[2+]/Zn[2+]SOD, G, SOD-1, SOD1, To, To-1, cSOD, l(3)108, l(3)68Af', l(3)G, DmelCG11793, CG11793

Reactivity: Plant

Application: ELISA

Certificates: ISO 9001:2008

Catalog no.: ABIN368404

Additional Information

Characteristics: This immunoassay kit allows for the in vitro rapid detection of Plant SOD concentrations in serum, plasma and other biological fluids. 

Alternative name: super oxide dismutase (SOD)

Sample Type: Serum, Plasma

Description: The enzyme superoxide dismutase (SOD), catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide. As such, it is an important antioxidant defense in nearly all cells exposed to oxygen. One of the exceedingly rare exceptions is Lactobacillus plantarum and related lactobacilli, which use a different mechanism. Several common forms of SOD exist: they are proteins cofactored with copper and zinc, or manganese, iron, or nickel. The cytosols of virtually all eukaryotic cells contain an SOD enzyme with copper and zinc (Cu-Zn-SOD). The Cu-Zn enzyme is a homodimer of molecular weight 32,500. The two subunits are joined primarily by hydrophobic and electrostatic interactions. The ligands of copper and zinc are histidine side chains. Chicken liver (and nearly all other) mitochondria, and many bacteria contain a form with manganese (Mn-SOD). The ligands of the manganese ions are 3 histidine side chains, an aspartate side chain and a water molecule or hydroxy ligand depending on the Mn oxidation state (respectively II and III). E. coli and many other bacteria also contain a form of the enzyme with iron (Fe-SOD), some bacteria contain Fe-SOD, others Mn-SOD, and some contain both. Fe-SOD can be found in the plastids of plants. The 3 active sites of Mn and Fe superoxide dismutases contain the same type of amino acid side chains. Simply stated, SOD outcompetes damaging reactions of superoxide, thus protecting the cell from superoxide toxicity. The reaction of superoxide with non-radicals is spin forbidden. In biological systems, this means its main reactions are with itself or with another biological radical such as nitric oxide (NO). The superoxide anion radical (O2-) spontaneously dismutes to O 2 and hydrogen peroxide (H 2 O 2 ) quite rapidly. SOD is biologically necessary because superoxide reacts even faster with certain targets such as NO radical, which makes peroxynitrite. Similarly, the dismutation rate is second order with respect to initial superoxide concentration. Thus, the half-life of superoxide, although very short at high concentrations is actually quite long at low concentrations. In contrast, the reaction of superoxide with SOD is first order with respect to superoxide concentration. Moreover, superoxide dismutase has the fastest turnover number of any known enzyme, this reaction being only limited by the frequency of collision between itself and superoxide.

Specificity: This assay recognizes plant SOD. No significant cross-reactivity or interference was observed.

Sensitivity: The minimum detectable dose of plant SOD is typically less than 0.125IU/ml . The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. Detection Range: The minimum detectable dose of plant SOD is typically less than 0.125IU/ml . The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero.

Synonyms: CG8905, DmelCG8905, dSOD2, Mn SOD, Mn-SOD, Mn-SOD2, mnSOD, MnSOD, MnSODII, Mn[2+]SOD, SOD, Sod-2, SOD-2, SOD2, Cu, Zn Sod, Zn-SOD, Cu-Zn SOD, Cu/Zn SOD, Cu/Zn superoxide dismutase, Cu/ZnSOD, CuSOD, CuZn SOD, CuZn-SOD, CuZnSOD, Cu[2+]/Zn[2+]SOD, G, SOD-1, SOD1, To, To-1, cSOD, l(3)108, l(3)68Af', l(3)G, DmelCG11793, CG11793

Application Details

Principle: The microtiter plate provided in this kit has been pre-coated with goat-anti-rabbit antibody. Standards or samples are then added to the appropriate microtiter plate wells with a Horseradish Peroxidase (HRP)-conjugated SOD and antibody preparation specific for SOD, and incubated. Then substrate solutions are added to each well. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of SOD in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Protocol: Reagent Preparation:
1. Bring all reagents to room temperature before use. 2. Wash Buffer If crystals have formed in the concentrate, warm up to room temperature and mix gently until the crystals have completely dissolved. Dilute 15 ml of Wash Buffer Concentrate into deionized or distilled water to prepare 300 ml of Wash Buffer. 3. Lysis Buffer Dilute 15 ml of Lysis Buffer Concentrate into deionized or distilled water to prepare 150 ml of 1X Lysis Buffer. 5.
Assay Procedure:
Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate. 6 1. Set a Blank without any solution. Add 50μl of Standard or Sample per well. 2. Add 50μl of HRP-Conjugate and 50μl of Antibody to each well. Not to Blank well! 3. Cover with the adhesive strip. Incubate for 1 hour at 37° C. 4. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (200μl) using a squirt bottle, multi -channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels. 5. Add 50μl of Substrate A and 50μl of Substrate B to each well. Incubate for 15 minutes at 37°C. Keeping the plate away from drafts and other temperature fluctuations in the dark. 6. Add 50μ l of Stop Solution to each well when the first four wells containing the highest concentration of standards develop obvious blue color. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. 7. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm. 7.
Calculation of Results:
Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the SOD concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
Limitations of the Procedure:
The kit should not be used beyond the expiration date on the kit label.Do not mix or substitute reagents with those from other lots or sources. 8It is important that the Calibrator Diluent selected for the standard curve be consistent with the samples being assayed.If samples generate values higher than the highest standard, dilute the samples with the appropriate Calibrator Diluent and repeat the assay.Any variation in operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cause variation in binding.This assay is designed to eliminate interference by soluble receptors, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.
Sample collection and storage:
Clean and grind plant tissues. Add 1mg plant tissues to 1ml Lysis Buffer. Put them on ice for a half-hour. Then homogenized them with a homogenizer, or with a ultrasonic disrupter until there is no significant tissue mass. Then after 10000rpm centrifugation for 5min, get the supernatant to do a pretest(50ul for each well). According to the result of pretest, determine whether it is needed for dilution with the PBS or ddH2O.

Assay Procedure: Assay Time: 1-3h
Sample Volume: 50-100ul

Application Notes: Detection Wavelength: 450 nm

Components: Reagent (Quantity): Assay plate (1), Standard (5x0.5ml), HRP-Conjugate (1x6ml), Antibody (1x6ml), Wash Buffer (20×concentrate) (1x15ml), Substrate A (1x7ml), Substrate B (1x7ml), Stop Solution (1x7ml), Lysis Buffer 1x15ml (10×concentrate)

Material not included: Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm. Pipettes and pipette tips. Deionized or distilled water. Squirt bottle, manifold dispenser, or automated microplate washer. Homogenizer or ultrasonic disrupter

Storage: 1. Unopened test kits should be stored at 2-8 C upon receipt and the microtiter plate should be kept in a sealed bag. The test kit may be used throughout the expiration date of the kit. Refer to the package label for the expiration date. 2. Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above. 3. A microtiter plate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.

Restrictions: For Research Use only