Lipid Peroxidation, Colorimetric Assay Kit, BioAssay™

Brand

BioAssay™

Kit Type

Assay

Tests

100

Sensitivity

0.1nmol/ml

Detection Method

Colorimetric

Sample Matrix

Tissue homogenates, cell culture, plasma and serum

EU Commodity Code

38220000

Shipping Temp

Blue Ice

Storage Temp

4°C

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.

References

1. Esterbauer, H., Schaur, R.J. and Zollner, H. (1991) Chemistry and Biochemistry of 4-Hyroxynonenal, Malonaldehyde and Related Aldehydes; Free Rad. Biol. Med. 11:81-128.2. Botsoglou, N.A. et al (1994) Rapid, Sensitive, and Specific Thiobarbituric Acid Method for Measuring Lipid Peroxidation in Animal Tissue, Food and Feedstuff Samples; J. Agric. Food Chem. 42:1931-1937.3. Carbonneau, M.A. et al (1991) Free and Bound Malondialdehyde Measured as Thiobarbituric Acid Adduct by HPLC in Serum and Plasma; Clin. Chem. 37:1423-1429.4. Bull, A.W. and Marnett, L.J. (1985) Determination of Malondialdehyde by Ion-Pairing High-Performance Liquid Chromatography; Analyt. Biochem. 149:284-290.5. Liu, J. et al (1997) Assay of Aldehydes from Lipid Peroxidation: Gas Chromatography-Mass Spectrometry Compared to Thiobarbituric Acid; Analyt. Biochem. 245: 161-166.

USBio References

No references available

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L2496-30 Lipid Peroxidation, Colorimetric Assay Kit, BioAssay™

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Tests

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