FRAP™ (Ferric Reducing Antioxidant Power) Detection Kit

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Purpose The DetectX® Ferric Reducing Ability of Plasma (FRAP™) Assay Kit is designed to quantitatively measure antioxidant status in a variety of samples. The assay measures the antioxidant ability from all species.
Brand FRAP™
Sample Type Serum, Plasma, Urine, Beverages, Juice, Cell Lysate, Plant Tissue, Fruit
Components Clear 96 well Half Area Plates 2 Plates Corning Costar Plate 3695
Ferrous Chloride Standard 90 μL Ferrous chloride at 10 mM in stabilizing solution
Assay Buffer Concentrate 25 mL A 10X acetate buffer concentrate with stabilizers and preservatives
Ascorbic Acid Control 1 Vial A vial containing 100 nmol Ascorbic Acid
FRAP Reagent A 1.4 mL FRAP Reagent A solution FRAP Reagent B 1.4 mL FRAP Reagent B solution
Material not included Deionized water.
Repeater pipet with disposable tips capable of dispensing 75 μL. 96 well plate reader capable of reading optical absorption at 560 nm.
Software for converting optical density (OD) readings from the plate reader and carrying out four parameter logistic curve (4PLC) fitting.
Background Reactive oxygen species (ROS)1,2 are produced as a consequence of normal aerobic metabolism3. These ''free radicals'' (FR) are usually removed or converted into other products in vivo by an ar- ray of antioxidants. Antioxidants are typically chemically stable atoms and molecules, which have one (or rarely more) free electron/electrons in their electron envelope. Almost all biomolecules, but mainly biomembranes, proteins and nucleic acids, may be attacked by reactive free radicals. Free radicals are responsible for many pathological processes, or they can be generated as the result of the pathological stage and cause important secondary damage to biological systems and cells1-5. Connections between free radicals and some serious diseases, including Parkinson's and Alzheimer's diseases, atherosclerosis, myocardial infarction, and chronic fatigue syndrome, have been demonstrated. However, short-term oxidative stress, the unbalance between the formation and scavenging of the reactive oxygen species, may be important in the prevention of aging due to triggering the process known as mitohormesis. On the average, 65-70 % of the population is excessively impacted by oxidative stress caused by FRs. In 1996 Iris Benzie and Sean Strain pub- lished a simple assay to measure antioxidant power6. The original demonstration of the power of this assay to measure antioxidant potential in serum and plasma has been extended to the anti- oxidant power of certain foods7, teas8, and fungi. The protective system of organisms is based on the activity of specific enzymes (especially su- peroxide dismutase, glutathione peroxidase, catalase, glutathione reductase) as well as non-enzy- matic compounds with antioxidant activity (ß-tocopherol, L-ascorbic acid, glutathione, coenzyme Q10, flavonoids, albumin and other molecules). Excess production of reactive oxygen species can also lead to inflammation, premature aging disorders, and several disease states, including cancer, diabetes, and atherosclerosis. Organisms have developed complex antioxidant systems to protect themselves from oxidative stress, however, excess ROS can overwhelm the systems and cause severe damage
Application Notes Optimal working dilution should be determined by the investigator.
Protocol A Ferrous Chloride standard is provided to generate a standard curve for the assay and all samples should be read off of the standard curve.
Samples are diluted in the provided Assay Buffer and added to the wells.
The FRAP Color Solution is made by mixing Reagent A and B with Assay Buffer.
The FRAP Color Solution is added to all wells and the plate incubated at room temperature.
Antioxi- dant power in the samples reacts with the FRAP Color Solution to generate a blue colored prod- uct which is read at 560 nm.
Reagent Preparation

Assay Buffer Dilute Assay Buffer Concentrate 1:10 by adding one part of the concentrate to nine parts of deion- ized water.
Once diluted this is stable for 3 months at 4 °C.
Ascorbic Acid Control 400 μL of diluted Assay Buffer should be added to the vial and vortexed.
The vial should be mixed for 5 minutes.
Unused reconstituted control solution should be aliquoted at 50 μL per vial to mini- mize freeze thaw cycles and stored at -20 °C. ® 6 WEB INSERT 150327 reagent preparatiOn (cOntinued) Standard Preparation Standards are prepared by labeling 6 tubes.
Add 180 μL of diluted Assay Buffer to the first tube and pipet 100 μL of diluted Assay Buffer into the rest of the tubes.
Carefully add 20 μL of the Fer- rous Chloride Standard Stock to the first tube and vortex.
Add 100 μL of the first tube to the sec- ond tube and vortex completely.
Repeat this for rest of the tubes.
The concentration of Ferrous Chloride in the tubes will be 1,000, 500, 250, 125, 62.5, and 31.25 μM FeCl2.
Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Assay Buffer (μL) 180 100 100 100 100 100 Addition Stock Std 1 Std 2 Std 3 Std 4 Std 5 Volume of Addition (μL) 20 100 100 100 100 100 FeCl2 Conc (μM) 1,000 500 250 125 62.5 31.25 Use all Standards within 2 hours of preparation.

Assay Procedure

Use the plate layout sheet on the back page to aid in proper sample and standard identification.
1. Pipet 20 μL of samples or standards into duplicate wells in the plate.
2. Pipet 20 μL of diluted Assay Buffer into duplicate wells as the Zero standard.
3. Pipet 20 μL of diluted Ascorbic Acid Control into duplicate wells as an optional control.
4. Add 75 μL of the prepared FRAP Color Solution to each well using a repeater pipet.
5. Incubate at room temperature for 30 minutes.
6. Read the optical density at 560 nm.

Calculation of Results

Average the duplicate OD readings for each standard and sample.
Create a standard curve by reducing the data using computer software capable of generating a four-parameter logistic curve (4PLC) fit.
The sample concentrations obtained should be multiplied by the dilution factor to obtain neat sample values.
Or use the online tool from power)-detection-kit.assay to calculate the data. *The MyAssays logo is a registered trademark of MyAssays Ltd. typical data Sample Mean OD Net OD FeCl2 Concentration (μM) Standard 1 2.021 1.905 1,000 Standard 2 1.092 0.976 500 Standard 3 0.604 0.488 250 Standard 4 0.352 0.236 125 Standard 5 0.244 0.128 62.5 Standard 6 0.177 0.061 31.25 Zero 0.116 0.0 0 Sample 1 1.976 1.860 974.3 Sample 2 0.553 0.437 225.0 Always run your own standard curves for calculation of results.
Do not use this data.
Ascorbic Acid Control This assay measures the ability of antioxidants to convert ferric to ferrous ions.
The ascorbic acid control indicates that the FRAP Color Solution is producing acceptable color reaction in response to a typical antioxidant.
Typical optical densities should be about 50 % of those produced by 1,000 μM FeCl2 standard. ® 9 WEB INSERT 150327 typical Standard curve 2.2 2.0 1.8 1.6 1.4 1.2 1a0n 0.8 0.6 0.4 0.2 0.0 0 200 400 600 800 1000 Ferrous Chloride Conc. (μM) Always run your own standard curves for calculation of results.
Do not use these data.

Assay Precision Three human samples diluted in Assay Buffer were run in replicates of 20 in an assay.
Inter Assay Precision:
Three human samples diluted in Assay Buffer were run in duplicates in eighteen assays run over multiple days by three operators. Kinetics of Color Development The FRAP assay chemistry is not a true end-point method. Color formation for some samples such as standards and diluted samples is almost instantaneous. Final color for some samples such as serum and plasma continues to develop however the rate of continued color development is relatively slow.
Restrictions For Research Use only
Preservative Sodium azide
Precaution of Use As with all such products, this kit should only be used by qualified personnel who have had labo- ratory safety instruction.
The complete insert should be read and understood before attempting to use the product.
Solutions containing sodium azide will yield an instantaneous colored product with the FRAP Color Solution.
No buffers or solutions containing azide can be measured using this kit.
Storage 4 °C
Storage Comment All components of this kit should be stored at 4°C until the expiration date of the kit.
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Product cited in: Sanguigni, Manco, Sorge, Gnessi, Francomano: "Natural antioxidant ice cream acutely reduces oxidative stress and improves vascular function and physical performance in healthy individuals." in: Nutrition (Burbank, Los Angeles County, Calif.), Vol. 33, pp. 225-233, 2016 (PubMed).

Zhang, Saremi, Gilbert, Wong: "Physiological and biochemical aspects of methionine isomers and a methionine analogue in broilers." in: Poultry science, Vol. 96, Issue 2, pp. 425-439, 2016 (PubMed).

Wang, Wang, Liu, Che, Xu, E: "Age-related variations of protein carbonyls in human saliva and plasma: is saliva protein carbonyls an alternative biomarker of aging?" in: Age (Dordrecht, Netherlands), Vol. 37, Issue 3, pp. 9781, 2015 (PubMed).

Mistry, Gill, Kurlak, Seed, Hesketh, Méplan, Schomburg, Chappell, Morgan, Poston et al.: "Association between maternal micronutrient status, oxidative stress, and common genetic variants in antioxidant enzymes at 15 weeks? gestation in nulliparous women who subsequently develop ..." in: Free radical biology & medicine, Vol. 78, pp. 147-55, 2014 (PubMed).