Cardiovascular and Oxidative Stress Assay Kit

TBARS Fluorometric Assay Kit

$345.00

The TBARS Fluorometric Assay Kit is intended for the quantitative determination of TBARS in serum, plasma, or urine by fluorometric assay method. 2-Thiobarbituric Acid Reactive Substances (TBARS) Fluorometric Assay kit is for research use only and not to be used in diagnostic procedures.

SKU: TBF39-K01 Categories: , ,

TBARS Fluorometric Assay Kit

The TBARS Fluorometric Assay Kit is For Research Use Only

Size: 1×96 wells
Sensitivity: 0.5 µM
Standard Range: 0.5 – 20 µM
Incubation Time: 30 minutes
Sample Type: Serum, Plasma, Urine
Sample Size: 100 µl

Assay Background

2-ThioBarbituric Acid Reactive Substances (TBARS) are naturally present in biological specimens and include lipid hydroperoxides and aldehydes which increase in concentration as a response to oxidative stress.1,2 TBARS assay values are usually reported in malonaldehyde (malondialdehyde, MDA) equivalents, a compound that results from the decomposition of polyunsaturated fatty acid lipid peroxides. The TBARS assay is a well-recognized, established method for quantifying these lipid peroxides, although it has been criticized for its reactivity towards other compounds other than MDA.3 This kit offers the researcher a straightforward, reproducible and consistent method for analyzing urine for lipid peroxidation products.

SAMPLE PREPARATION
When working with plasma, the sample should be deproteinated with an acid. Centrifuge and use the supernatant to perform the assay. This solution may appear cloudy after the reaction, and can be clarified by passing through a 0.2 µ syringe filter. When working with urine, colored compounds contribute to the signal measured at 532 nm. This interference can be removed by running a sample blank with each sample.
Urine:
1. Urine samples can be used directly and should be assayed immediately. If the assay is to be performed on a different day, the sample should be stored at -70°C.

Plasma and Serum:
1. Collect blood samples and process immediately per the collection tube instructions
2. Prepare a saturated solution of ammonium sulfate.
3. Add 100 µl of saturated ammonium sulfate to 0.5 mL of serum or plasma in a test tube or microcentrifuge tube.
4. Add 35 mg TCA (trichloroacetic acid) to each sample and vortex. A cloudy precipitate should form.
5. Centrifuge the tubes and transfer the supernatant to a clean tube. Plasma and serum samples can be run without dilution. Samples are now ready for assay.

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TBARS ELISA Assay Kit
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Additional Information

Assay Principle

This TBARS Fluorometric Assay Kit is based on the reaction of a chromogenic reagent, 2-thiobarbituric acid, with MDA at 25°C. One molecule of MDA reacts with 2 molecules of 2-thiobarbituric acid via a Knoevenagel-type condensation to yield a chromophore with absorbance maximum at 532 nm, as shown below in Figure 1:

Free MDA

  1. Preparation of Standards and Samples:  Add each of the following reagents into microcentrifuge tubes and mix well.
    • Standards: 100 μL of standard and 300 μL of Indicator Solution.
    • Samples: 100 μL of sample and 300 μL of Indicator Solution.
    • Blanks: 100 μL of sample and 300 μL of Acid Reagent.
  2. After the standards, samples and blanks have been mixed; allow them to react for 20 minutes at room temperature.
  3. Transfer 150 µL of each solution to the microplate and read at 540 nm. Please note that the optimum absorbance wavelength for this assay is 532 nm. The pink color is stable for several hours at room temperature.

Total MDA

Prepare samples and standards exactly as above, but heat sample at 65C for 30 minutes, then follow step 3 as above.

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Literature References

  1. Armstrong, D.; Browne, R. (1994) Free Rad. Diag. Med. 366; 43-58.
  2. Botsoglou, N.A., et al.; (1994) J. Agric. Food Chem. 42; 1931-1937.
  3. Yagi, K. (1998) Free Rad. Antiox. Prot., 108; 101-106.
  4. Moore, K., Roberts, L.J., (1998) Free Rad. Res. 28; 659-671.
  5. Liu, J., et al.; (1997) Analyt. Biochem. 245;161-166.
  6. Carbonneau, M.A., et al.; (1991) Clin. Chem. 37; 1423-1429.
  7. Mattson, J.P., et al.; (2002) Pathophysiology 8; 215-221.
  8. Calvo, J.R., et al.; (2001) J. Cell Biochem. 80; 461-70.
  9. Janciauskiene, S. and Bo Ahren; (2000) Biochem. Biophys. Res. Com. 267; 619-625.
  10. Montilla-Lopez, P., et al.; (2002) European J. Pharmacology 451; 237-243.

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