Lipid Peroxidation, Colorimetric Assay Kit, BioAssay™

Lipid peroxidation is a well-established mechanism of cellular injury in both plants and animals, and is used as an indicator of oxidative stress in cells and tissues. Lipid peroxides are unstable and decompose to form a complex series of compounds including reactive carbonyl compounds. Polyunsaturated fatty acid peroxides generate malondialdehyde (MDA) and 4-hydroxyalkenals (HAE) upon decomposition. The measurement of MDA has been used as an indicator of lipid peroxidation. This kit is designed to measure MDA.||Assay Principle:|This assay is based on the reaction of a chromogenic reagent, N-methyl-2-phenylindole (R1), with MDA at 45°C. One molecule of MDA reacts with 2 molecules of reagent R1 to yield a stable chromophore with maximal absorbance at 586nm.||Kit Components:|L2496-30A: Reagent R1 ( N-methyl-2-phenylindole in acetonitrile), 3 x18ml|L2496-30B: MDA Standard (1,1,3,3-Tetramethoxypropane in Tris-HCl ), 1x1ml|L2496-30C: Diluent (Ferric Iron in Methanol), 1x30ml||Storage and Stability:|Store kit components at 4°C. Kit is stable for 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. ||Materials Required But Not Supplied:|1. Spectrophotometer for measuring absorbance at 586nm|2. Spectrophotometric cuvettes with a 1cm optical path length|3. Water bath at 45°C.|4. Disposable glass test tubes and stoppers compatible with acetonitrile, methanol and acid.|5. 37% HCl|6. Butylated hydroxytoluene (BHT)|7. Acetonitrile|8. Microcentrifuge|9. Polypropylene microcentrifuge tubes|10. Deionized water|11. Adjustable micropipettes (10-100ul) and tips||Assay Procedure:|1. Add 200ul of Standards or Samples to a microcentrifuge tube.|2. Add 650ul of diluted Reagent R1 to each tube and vortex.|3. Add 150ul 37% (12N HCl) to each tube and mix well.|4. Incubate at 45°C for 60 minutes.|5. Centrifuge samples at 15,000 x g for 10 minutes to obtain a clear supernatant.|6. Transfer the supernatant to a cuvette and read the absorbance at 596nm.