TBARS assay - (Jun/03/2010 )

Hello all!

I've been searching info concerning the thiobarbituric acid reactive substances assay to detect lipoperoxide levels on seaweeds, but I have some doubts concerning the protocols I find, specially when it comes to the over-the-bench craftiness taht usually is not described in articles. May I add that I have no experience whatsoever with these kind of assays.

Would anyone experienced be so kind to help me by providing me with a clear protocol and advice for this assay, please?

Thank you!

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A escutar: Manu Chao - Clandestino
via FoxyTunes

zeptometrix sells a tbars assay kit (here is the pdf of the instructions).

zeptometrix oxidative stress products

there is a conventional method also to measure LPO. ref. Beuge and Aust 1978.

here's a method for you:

Lipid peroxidation: Malondialdehyde (MDA), the by-product of L.P.O forms adduct with TBA. On boiling, it produces pink colored complex, which absorbs maximally at 532 nm.

Reagents:
1. Tris-HCl buffer: 150mM (pH7.1)
2. Ascorbic acid: 1.5 mM
3. Ferrous sulphate: 1.0 mM
4. TCA (10%)
5. TBA: 0.375g%

Procedure: 0.1 ml sample, 0.1 ml Tris-HCl buffer, 0.1 ml FeSO4 and 0.1 ml Ascorbic acid were added in a test tube then 0.6 ml DDW was added to make the volume 1.0 ml. It was incubated at 370C for 15 min. Then 1.0 ml TCA and 2 ml TBA were added to the reaction mix. Tubes were plugged and incubated for 15 min. at boiling water. Centrifugation was done at 3000 rpm for 10 min. Readings were taken at 532 nm.

Calculations:
The concentration of MDA is calculated using extinction coefficient of MDA-TBA complex which is 1.56 × 105 M-1 cm-1 and the resuts are expressed as nmoles MDA/mg protein.

formula: OD × sample volume
1.56×105×total volume × mg protein/ml