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Product Details
| Cat # +Size | K671-100 |
|---|---|
| Size | 100 assays |
| Detection Method | Absorbance (545-600 nm) |
| Species Reactivity | Mammalian |
| Applications | The assay can detect 0.2 to 20 nmol of ascorbic acid in various samples. |
| Features & Benefits | • Simple procedure; takes ~ 30 minutes to complete the assay • Fast and convenient |
| Kit Components | • FRASC Buffer • Ascorbic Acid Probe • FeCl3 solution • Ascorbate Oxidase (lyophilized) • Ascorbic Acid Standard (20 µmole) |
| Storage Conditions | -20°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
Ascorbic Acid (Vitamin C) plays an important role in many biological processes. It is a potent anti-oxidant, anti-inflammatory, anti-viral agent and an immune stimulant and is present in a wide variety of biological specimens. Due to the presence of a variety of other antioxidants in biological samples such as serum, most ascorbic acid assays show strong interference. BioVision’s FRASC Assay Kit provides a rapid, simple, and sensitive means of detecting ascorbic acid in biological samples such as serum and other body fluids, tissue and cell extracts, growth media and food products. In this assay, Feᶟᶧ is reduced to Fe²ᶧ by any antioxidants present. The ferrous iron is chelated with a colorimetric probe to produce a product with a strong absorbance band which can be monitored between 545-600 nm. The addition of ascorbate oxidase to parallel samples removes any ascorbate present leaving a background value which is subtracted from the total to give ascorbate content. The assay can detect 0.2 to 20 nmol of ascorbic acid in various samples.
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Would your kits K661-100 or K671-100 be suitable for measuring ascorbic acid levels in blackcurrant juice? The juice itself has quite a strong colour. Even with diluting it down, would you know if one of these 2 kits could be suitable?
K661 and K671 measurements are in the yellow-orange range of the spectrum (545-600nm). There could be some interference from the blackcurrant juice in this range but you can run parallel controls and subtract the background to get the final numbers for your assay. So either one of these could work. K661 allows both colorimetric and fluorometric measurements and K671 allows only colorimetric measurement. I would recommend not using the fluorometric version of the assay since it is 10-100 fold more sensitive and the background color intereference may be more pronounced in this case.
Could results be low depnding on how the samples are prepared?
Ascorbic acid is unstable and very easily oxidizable, especially if there is time lag in preparing samples for the assay. It is essential to work quickly and keep sample vials closed as much as possible to prevent air-exposure. This could be the reason for low amounts of unoxidized, free ascorbic acid detected in the samples.
The OD values are beyond the std curve highest point. How can I calculate amount of Ascorbic acid from this Std. curve?
If the OD values from your samples are higher than the linear range of the assay, then you might not get the expected values. There will be underestimation because the std. curve can only correctly predict calculations within the linear range. You might need to dilute the samples and then try again. We recommend taking one or two representative samples and using different volumes to make sure the values are within the linear range of the std. curve.
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. It is recommended 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.
Are trial sizes of this kit available?
Unfortunately, we do not have trial sizes of this kit available. However,for customers based in the US or Canada, a 10% off list price introductory discount can be offered on the list price. For all other customers based out of this area our regional BioVision distributor should be contacted.
Is it essential to make a standard curve for every expt, or is one curve/kit enough?
Yes, it is strongly 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.
Can alternate buffers be used 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.
Can individual components of this kit be purchased separately?
Yes, any of the kit's components can be purchased separately without having to buy the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
What is the sample preparation protocol for cells and tissues?
Cells (1X106 in 100 ul) and tissues (10 mg in 100 ul) can be homogenized in water by using a dounce homogenizer or sonicator on ice until the tissue is lysed completely. Once the homogenization is completed, centrifuge at 10,000g for 15 min. Take the supernatant and use supernatant for the assay. If there are more tissue/cells than the suggested amounts, you can increase the amount of water.
What does this kit detect? Ascorbic acid or dehydroascorbic acid?
The kit detects ascorbate/ascorbic acid and not the dehydroascorbic acid.
Sage et al., Human erythrocytes transport dehydroascorbic acid and sugars using the same transporter complex. Am J Physiol Cell Physiol, May 2014; 306: C910 - C917.
Theodorou, A. A. et. al. No effect of antioxidant supplementation on muscle performance and blood redox status adaptations to eccentric training. Am J Clin Nutr, Jun 2011; 93: 1373 - 1383.
For more citations of this product click here
