TBARS assay kit and TBARS assay protocol

The TBARS assay (thiobarbituric acid reactive substance assay) is used to measure lipid peroxidation in cell and tissue extracts, and biological fluids.

Learn more about the TBARS assay method here, review our protocol to set up the assay yourself, or use our TBARS assay kit.

TBARS assay method

The TBARS assay detects the level of MDA (malondialdehyde), the major lipid oxidation product, and also some minor related compounds.

It is often considered a good index of the level of oxidative stress in a biological sample.

In the assay, thiobarbituric acid reacts with MDA to form a red product which can be detected using a colorimetric (OD = 532 nm) or fluorometric (Ex/Em 532/553 nm) plate reader.

TBARS assay kit

For robust results with your TBARS assay, we recommend Lipid Peroxidation (MDA) Assay Kit (ab118970) or alternative MDA assay kit ab233471. This is supplied with a full optimized protocol and the critical reagents for your assay, including an MDA standard to enable quantitation.

TBARS assay protocol

A simplified protocol for the TBARS assay, and tips for success, is as follows.

1. Reconstitute TBA in glacial acetic acid (use only glacial acetic acid, as regular acetic acid affects TBA stability due to its high-water content). The reconstituted solution is typically stable for 1 week at +4°C or -20°C. Sonicate in a RT water bath if required to dissolve precipitates.

2. Use BHT in your lysis buffer during sample preparation to stop further sample peroxidation while processing. For plasma/serum samples, use phosphotungstic acid to precipitate the lipids for analysis.

3. To assay the samples, add TBA solution to your prepared samples. Incubate at 95°C for 60 minutes. Cool to room temperature in an ice bath for 10 minutes.

4. The sensitivity of the TBARS assay can be improved by using n-butanol to precipitate the MDA-TBA adduct after step 3, then dissolving the precipitate in water after removal of n-butanol.

5. Measure the output immediately after processing the samples, on a microplate reader at OD 532 nm for a colorimetric assay,  and at Ex/Em = 532/553 nm for a fluorometric assay.

For instructions on data analysis, and a full experimental protocol – consult our Lipid Peroxidation Assay Kit (ab118970) or alternative MDA assay kit ab233471.

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