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Product Details
| Cat # +Size | K274-100 |
|---|---|
| Size | 100 assays |
| Detection Method | Absorbance (570 nm) |
| Species Reactivity | Mammalian |
| Applications | Assay measuring either the combination of both small molecule and protein antioxidants or small molecules antioxidant alone in the presence of our proprietary Protein Mask. |
| Features & Benefits | • Simple procedure; takes less than 2 hours • Fast and convenient |
| Kit Components | • Cu²ᶧ Reagent • Assay Diluent • Protein Mask • Trolox Standard |
| Storage Conditions | 4°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
Antioxidants play an important role in preventing the formation of and scavenging of free radicals and other potentially toxic oxidizing species. There are three categories of antioxidant species: enzyme systems (GSH reductase, catalase, peroxidase, etc.), small molecules (ascorbate, uric acid, GSH, vitamin E, etc.) and proteins (albumin, transferrin, etc.). Different antioxidants vary in their reducing power. Trolox is used to standardize antioxidants, with all other antioxidants being measured in Trolox equivalents. Measurement of the combined nonenzymatic antioxidant capacity of biological fluids and other samples provides an indication of the overall capability to counteract reactive oxygen species (ROS), resist oxidative damage and combat oxidative stress-related diseases. In some cases, the antioxidant contribution of proteins is desired whereas in other cases only the contribution of the small molecule antioxidants is needed. BioVision developed the TAC Assay Kit, which can measure either the combination of both small molecule antioxidants and proteins or small molecules alone in the presence of our proprietary Protein Mask. Cu²ᶧ ion is converted to Cu+ by both small molecule and protein. The Protein Mask prevents Cu²ᶧ reduction by protein, enabling the analysis of only the small molecule antioxidants. The reduced Cu+ ion is chelated with a colorimetric probe giving a broad absorbance peak around 570 nm, proportional to the total antioxidant capacity.
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Why do we get different results from the same sample at different times?
This can occur due to 3 main possibilities:
1. Different lots may give different readings. However, when using calibration curve together with the sample testing, the sample concentration should be in the same range.
2. Sample may change during storage, especially Vitamin C.
3. When using the same lot, the same sample reading should be similar, otherwise maybe there was some experimental error.
1. Different lots may give different readings. However, when using calibration curve together with the sample testing, the sample concentration should be in the same range.
2. Sample may change during storage, especially Vitamin C.
3. When using the same lot, the same sample reading should be similar, otherwise maybe there was some experimental error.
Is the theory of TAC assay kit the same as the DPPH radical method? We are using DPPH reagent (final conc 250uM in toluene) and want to know is there any advantage for using your TAC assay kit.
The two assays are different from one another. Our TAC assay uses Cu++ to Cu+ as a mechanistic tool. It is easy to reduce so some molecules such as uric acid will respond quickly to it. Some of the disadvantages of the DPPH are:
1) This method quantifies DPPH after exposure to sample-The standard curve gives negative results.
2) Reaction kinetics between DPPH and anti oxidants are not linear. So the anti oxidant capacity is rather arbitrary.
3) The time to steady state using DPPH varies with different anti oxidants. So we may get conflicting relative capacity depending on the reaction time.
1) This method quantifies DPPH after exposure to sample-The standard curve gives negative results.
2) Reaction kinetics between DPPH and anti oxidants are not linear. So the anti oxidant capacity is rather arbitrary.
3) The time to steady state using DPPH varies with different anti oxidants. So we may get conflicting relative capacity depending on the reaction time.
Does EDTA in blood affects the use of this kit on plasma?
The presence of EDTA should have no problem on the function of this kit.
Is the protein mask necessary with urine sample, since the level of protein in urine compared to serum is minimal?
If the total antioxidant capacity is desired, I would not recommend the use of the protein mask. If only the levels of the small molecule antioxidants are required, please use the protein mask.
Which sample is better between serum and plasma? Why?
Both human serum and plasma have an antioxidant capacity of 0.5-2mM. So both samples would be equally good for detection.
To draw the standard curve, “Add 0, 4, 8, 12, 16, 20 µl of the Trolox standard to individual wells.Adjust the total volume to 100 µl with ddH2O to give 0, 4, 8, 12, 16, 20 nmol of Trolox standard.” It’s not serial dilution. That is ,they first make 32mM solution ,then they dilute 32mM solution to 16mM. then they use 16mM solution to make 8mM solution and so on. Do you think which method is better?
The standards we recommend are not serial dilutions. Please use them as recommended.
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 kit's 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 your 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 kit's 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, I would strongly recommend you 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.
Groner, Judith A. et al. (2016) Oxidative Stress in Youth and Adolescents With Elevated Body Mass Index Exposed to Secondhand Smoke Nicotine Tob Res. 2016 Jul;18(7):1622-7.
Waly et al., Low nourishment of B-vitamins is associated with hyperhomocysteinemia and oxidative stress in newly diagnosed cardiac patients. Experimental Biology and Medicine, Aug 2015; 10.1177/1535370215596860.
Capllonch-Amer et al., Estradiol stimulates mitochondrial biogenesis and adiponectin expression in skeletal muscle.J. Endocrinol., May 2014; 221: 391 - 403.
Karen M. Young et al., Each to their own: skeletal muscles of different function use different biochemical strategies during aestivation at high temperature. J. Exp. Biol., Mar 2013; 216: 1012 - 1024.
Hirai, D. et. al. Acute antioxidant supplementation and skeletal muscle vascular conductance in aged rats: role of exercise and fiber type. Am J Physiol Heart Circ Physiol, Apr 2011; 300: H1536 - H1544.
For more citations of this product click here
