• Detection method- Absorbance (450 nm)
• Sample type- Cell and Tissue lysates, culture media, urine, plasma and serum, as well as many other biological fluids
• Species reactivity- Mammalian
• Applications- Kit contains the necessary reagents for convenient measurement of activity of Superoxide Dismutase (SOD) by colorimetric method.
Features & Benefits:
• Simple one-step procedure; takes around than 30 minutes
• Fast and convenient
• WST Solution
• SOD Enzyme Solution
• SOD Assay Buffer
• SOD Dilution Buffer
Superoxide dismutase (SOD) is one of the most important antioxidative enzymes. It catalyzes the dismutation of the superoxide anion into hydrogen peroxide and molecular oxygen. The sensitive SOD assay kit utilizes WST-1 that produces a water-soluble formazan dye upon reduction with superoxide anion. The rate of the reduction with a superoxide anion is linearly related to the xanthine oxidase (XO) activity, and is inhibited by SOD (below). Therefore, the inhibition activity of SOD can be determined by a colorimetric method.
USAGE: For Research Use Only! Not For Use in Humans.
Our technicians are confused about how to run the kit as no standard is provided and nor is it mentioned as to which type of plate should be used. Do we have sod standard that we may purchase or let us know how we can prepare one. also, let us know which plate should we use?
The SOD kit (K335) can be used without a standard since it is reporting as %inhibition. Thus, the more SOD in a sample the less WST-1 formazan produced (which was dependent on Xanthine Oxidase (XO) activity). You can use the purified protein as a standard (Cat # 4802) and then treat the standard just as you would treat a sample (see protocol on attached data sheet). The 96-well plate is a standard flat bottom plate such as the one sold by Greiner Bio one (#655-101) or any other standard polystyrene plate.
How do we prepare cell/tissue samples to use with this kit?
Before treatment, tissue should be perfused with PBS or 150mM KCl to remove any red blood cells which may be contaminating the tissue. Homogenize tissue or lyse cells in ice cold 0.1M Tris/HCl, pH 7.4 containing 0.25M sucrose, 5mM β-ME, 0.1mg/ml PMSF.
Centrifuge crude tissue homogenate/cell lysate at 500 x g for 5 minutes at 4°C and discard the cell debris pellet. The supernatant contains cytosolic and mitochondrial enzyme activity. The mitochondria should be lysed by adding 50ul/ml of 10% Triton X-100. This preparation can be used for total SOD activity.
How do we differentiate between cytosolic and mitochondrial SODs?
To differentiate between cytosolic and mitochondrial SOD activity, use BioVision K256-100 Mitochondrial/Cytosol Fractionation Kit or manually separate the fractions-centrifuge the supernatant at 14,000 x g for 10 min at 4°C to pellet the mitochondria. Pour off the supernatant which contains the cytosolic enzyme activity and Store at -80°C until ready to use. Resuspend the mitochondrial pellet in more ice cold homogenization buffer and repellet at 14,000 x g for 10 min at 4°C. The pellet is purified mitochondria and should be kept on ice until used. The mitochondria can be lysed by suspending in PBS containing 0.5% Triton X-100. The suspension is ready to be analyzed and should be assayed for protein content to normalize enzyme activity.
How do we prepare blood samples to use with this kit?
Collect blood using citrate or EDTA. Centrifuge at 1,000 x g for 10 min at 4°C. Remove the plasma layer without disturbing the buffy layer and store at -80°C until ready for analysis. Remove the buffy layer from the red cell pellet. Resuspend the erythrocytes in 5x volume of ice cold distilled water and centrifuge at 10,000 x g to pellet the erythrocyte membranes. Store the supernatant at -80°C until ready for analysis. Plasma can be diluted approx. 3-10x and the red cell lysate diluted approx 100x prior to SOD assay.
What mass of tissue is recommended as starting material for this assay?
The minimum amount you can start with for this is ~10 mg.
Is there any formula of transforming relative enzyme activity into absolute enzyme activity? Or does it need a SOD standard to get the absolute activity of SOD?
Yes, you would need to use a standard to find out the absolute activity. Here is the recombinant protein you can use as the standard: http://www.biovision.com/superoxide-dismutase-human-recombinant-3644.html. You will have to do a kinetic experiment to get this absolute activity. I can give you an exapmple of how to calculate the activity:
Plot a standard curve with different conc of the SOD using exactly this assay protocol.
For all samples, do a kinetc assay: Measure OD 450 nm at T1 to read A1, measure again at T2 after incubating the reaction at 25°C for 10 - 20 min (or incubate longer time if the SOD activity is low in sample) to read A2, protect from light. The signal increase is due to SOD, ∆A = A2 – A1
Note: It is essential to read A1 and A2 in the reaction linear range. It will be more accurate if you read the reaction kinetics. Then choose A1 and A2 in the reaction linear range.
Calculation: Subtract 0 standard readings from the standards. Plot the SOD standard curve. Apply the ∆A to the standard curve to get B nmol of activity generated between T1 and T2 in the reaction wells. SOD calculation:
SOD Activity = [B/(T2-T1)xV] x sample dilution factor = nmol/min/ml = mU/ml.
Where: B is the SOD amount from SOD standard curve (in nmol).
T1 is the time of the first reading (A1) (in min).
T2 is the time of the second reading (A2) (in min).
V is the sample volume added into the reaction well (in ml).
Could you please advise me, should the protein concentration be measured of the samples tested? The protocol you have provided shows a graph of percent inhibition vs SOD activity, has this been normalised to the protein concentration?
The graph we have on our datasheet has the % inhibition on Y-axis. From this kit protocol, you will just get the Y-value which is the % inhibition. To draw the graph, you will need the X value. You can get that by using standards (not included in the kit). The standards (Cat # 4802-100) have to be treated the same way as the samples, but since you already know the amount you are taking, you can easily calculate U/ml.
Can this kit be used with samples like bacteria, plants, drosophila, yeast etc?
We have optimized the kit with mammalian samples. However, theoretically these kits should work with samples from multiple species/sources. Since the optimal conditions depend on the sample type, the protocol has to be be adapted to fit the samples for efficient results. Please refer to this kits citations to see what kind of samples have been used with this kit other than mammalian samples.
Can we use frozen samples with this assay?
Fresh samples are always preferred over frozen samples. However, frozen samples can also be used, provided, they were frozen right after isolation, were not freeze thawed multiple time (for which we recommend aliquoting the samples before freezing) and have been frozen for relatively short periods.
Can we use a different wavelength than recommended for the final analysis?
It is always recommended to use the exact recommended wavelength for the most efficient results. However, most plate readers have flexibility in their band width of detection in increments of +/- 10 nm. Depending on this flexibility range, you can deviate from the recommended wavelengths within limits.
What is the exact volume of sample required for this assay?
There is no specific volume we can recommend for the amount any sample to be used since it is completely sample concentration and quality based. You have to do a pilot expt with multiple sample volumes to determine the optimal volume which gives a reading within the linear range of the standard curve. Please refer to the citations for this product to see what other clients have used with similar sample types.
Do you have trial sizes of this kit?
Unfortunately, we do not have trial sizes of this kit available. However, if you are based in the US or Canada, we will give you a 10% off list price introductory discount on its purchase price. If you are based out of this area please contact yopur regional BioVision distributor.
What is the shelf life of this kit?
This kit is good for 12 months from the date of shipment in the unopened form when stored at the appropriate temperature and appropriate conditions. After opening and reconstitution, some of the components in this kit are good for 2 months at -20°C. Please refer to the datasheet for storage information and shelf life of each of the components.
Why are my standard curve values lower than those shown on the datasheet?
There are multiple factors which influence the signals like the incubation times, room temperature, handling etc. In general, to increase the value of the standards, you can increase the incubation time. As long as the standard curve is linear, it should be fine to use, since all of your samples will also be measured under the same conditions on this curve.
How do I normalize my samples against protein concentration?
You can use a protein quantitation assay on the supernatants you get from cell/tissue lysates or with any other liquid sample in the assay buffer.
Can we purchase individual components of this kit?
Yes, you can purchase any of the kits components without the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Can we use an alternate buffer for sample preparation (cell lysis, sample dilutions etc)?
Our assay buffers are optimized for the reactions they are designed for. They not only contain some detergents for efficient lysis of your cells/tissue, but also contain some proprietary components required for the further reactions. Therefore, we highly recommend using the buffers provided in the kit for the best results.
Should I make a standard curve for every expt I do, or is one curve/kit enough?
Yes, It is recommended to do the standards every time you do the expt. There is always a chance that something was done differently that day and we do not want any conditions to differ between standards and samples.