Antioxidant Assay Kit, BioAssay(TM)

An Antioxidant is a molecule capable of slowing or preventing the oxidation of other molecules. Antioxidants protect the cells from damages by reactive oxygen species which are produced in oxidation reactions in the cell. Antioxidants can be small molecules such as glutathione, vitamins, or macromolecules such as catalase, glutathione peroxidase. As oxidative stress contributes to the development of many diseases including Alzheimer's disease, Parkinson's disease, diabetes, rheumatoid arthritis and neurodegeneration, the use of antioxidants in pharmacology is intensively studied. Antioxidants are also widely used as dietary supplements and in industry as preservatives in food, cosmetics, rubber and gasoline.\nSimple, direct and high-throughput assays for total antioxidant capacity (TAC) find wide applications in research, food industry and drug discovery. The improved assay measures total antioxidant capacity in which Cu2+ is reduced by antioxidant to Cu+. The resulting Cu+ specifically forms a colored complex with a dye reagent. The color intensity at 570nm is proportional to TAC in the sample.\n\nKey Features:\nSensitive and accurate. Use 20ul sample. Linear detection range from 1.5 to 1000uM Trolox equivalents.\nSimple and high-throughput. The procedure involves addition of a single working reagent and incubation for 10 min. Can be readily automated as a high-throughput assay for thousands of samples per day.\n\nApplications:\nDirect Assays: serum, plasma, urine, saliva and other biological samples, food and beverages.\nDrug Discovery/Pharmacology: effects of drugs on TAC.\n\nKit Contents:\nReagent A: 12ml Reagent B: 1ml\nStandard: 100ul 50mM Trolox\nStorage conditions. The kit is shipped at room temperature. Store Reagent A at room temperature and other components at -20 degrees C. Shelf life of six months after receipt.\nPrecautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.\n\nSample Preparation:\nSamples should not contain any metal chelators (e.g. EDTA) and should be clear and free of any turbidity or particles. Liquid samples (e.g. nonhemolyzed serum, plasma) can be assayed directly. Cell lysate is prepared by homogenizing or sonicating cells in ice-cold 1 x PBS and centrifugation for 10 min at 14,000 rpm to pellet any debris. Use the clear supernatant for the assay. If not assayed immediately, freeze supernatant at -80 degrees C (stable for 1 month).\n\nAssay Procedure:\n1. Standards and Samples. Equilibrate all components to room temperature. Briefly centrifuge Reagent B and Standard before opening. Mix 5ul of the standard with 245ul dH2O (final 1mM Trolox). Dilute standards as shown in the Table below. Transfer 20ul standards into wells of a clear flat-bottom 96-well plate.\nNo 1mM Trolox+H2O Vol (ul) Trolox (uM)\n1 100ul+0ul 100 1000\n2 60ul+40ul 100 600\n3 30ul+70ul 100 300\n4 0ul+100ul 100 0\nTransfer 20ul of each sample into separate wells of the 96-well plate.\nNote: for unknown samples, perform several dilutions to ensure that TAC is within the linear range of 1.5 to 1000uM Trolox equivalents.\n2. Assay. Prepare enough Working Reagent for Sample and Standard wells by mixing, for each assay well, 100ul Reagent A and 8ul Reagent B. Add 100ul Working Reagent to all assay wells. Tap plate to mix. Incubate 10 min at room temperature.\n3. Read OD570nm on a plate reader.\nNote: if calculated TAC is higher than 1000uM Trolox equivalents, dilute sample in dH2O and repeat assay. Multiply the results by the dilution factor.\n\nCalculation:\nSubtract blank OD value (#4) from all standard and sample OD values. Plot the DOD570nm against standard concentrations and determine the slope of the standard curve. Calculate the Total Antioxidant Capacity (TAC) of Sample,\nTAC =\nODSAMPLE-ODBLANK\nSlope (uM-1)\nx n (uM Trolox Equivalents)\nODSAMPLE and ODBLANK are the OD570nm values of the sample and H2O blank (standard #4). n is the sample dilution factor.\n\nMaterials Required, But Not Provided:\nPipetting devices, centrifuge tubes, clear flat-bottom uncoated 96-well plates, plate reader capable of reading optical density at 570nm, homogenizer or sonicator etc.

Prices direct from United States Biological

Quick response times

Exclusive Biosave savings/discounts

SPECIFICATIONS

Catalog Number

A2298-44G

Size

1Kit

References

1. Sies H (1997). Oxidative stress: oxidants and antioxidants. Exp Physiol 82: 291-295.\n2. Cao G, Alessio H, Cutler R (1993). Oxygen-radical absorbance capacity assay for antioxidants. Free Radic Biol Med 14: 303-311.\n3. Prior R, Wu X, Schaich K (2005). Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J Agric Food Chem 53: 4290-4302.