I am getting different concentration values for the same sample. For liver tissue, at first I got 35 µM concentration. But, after one day the concentration value has come 13.5 µM.To assay MDA from tissue I have followed the protocol below-
For standard curved I have used 100 µM, 50 µM, 25 µM, 12.5 µM, 6.25 µM MDA dissolved in homogenizing buffer.
Every time I used to make standard curve prior to experiment. How should I troubleshoot my problem? Thanks in advance.
Dear Debaprasad
Your are following the TBARS assay which have low reproductibility and then need a strict control of operating conditions. I can just give you some hints:
1- Make sure all reagent are freshly prepared;
2- Use TCA 2.8%; Make sure the TBA is fully dissolved (if not prepare it in NaOH 0.05 M) and use a ratio TBA/TCA of 1/10 (v/v) for the assay;
3- Make a strict control of temperature in the water bath (95°C for example) as well as incubation time;
4- Run the standard and test samples simultaneously.
5- Repeat the procedure to be sure you have the same results.
Regards!
NOBOSSE
Dear Debrapasad,
Interesting information about procedures for lipid peroxidation measurement is available in the following link.
Finally, if you consider this answer appropriate, please upvote it using the green up arrow click.
Sincerely,
http://nopr.niscair.res.in/bitstream/123456789/3805/1/IBB%2040(5)%20300-308.pdf
Dear Debaprasad Koner,
I would recall the same issues suggested by Pierre, the only difference that in our lab a 20% TCA and therefore TCA/TBA ratios varied from the mentioned (~1/1) without problem. The issue with dissolving TBA deserves special attention, especially because it also changes the fluorescence levels depending on time and preparation procedure. In addition to that:
1- You could consider to add an antioxidant as BHT in your homogenates to prevent further MDA production during sample preparation.
2- You could add an emulsification solution as SDS to the assay to improve reaction rates. (If your samples contain relevant fat amounts I would also dilute them and measure by fluorescence instead).
3- You could consider a final extraction with a solvent as N-butanol to measure absorbance/ fluorescence only in the fraction really reactive of your assay.
Please, find attached a protocol which we have been using for a while with high reproductibility.
All the Best
Abel
Uchiyama, M., Mihara, M., 1978.Determination of malonaldehyde precursor in tissues by thiobituric acid test, Anal.Biochem. 86, 271-278.
Your problem was getting different values that because storing tissue homogenate. Thus use fresh homogenate remaining all above what remaining suggested in that any method u can use ie using TCA and TBA.
HPLC is better and accurate,, also can be assayed using spectrophotometry by reaction with TBA ,, but it is not accurate
I worked with the following colourimetric procedure which gave me good results by the end of my research however i worked with commercial available kit that was faster and more accurate good luck
- Small parts of livers were cut, weighed and homogenized in 9 volumes of 1.15% KCl to prepare 10% homogenate (w/v).
- An aliquot of 0.1 ml liver homogenate was added to 0.2 ml of SDS. This was followed by the addition of 1.5 ml of acetic acid and 1.5 ml of aqueous solution of TBA. This mixture was finally made up to 4 ml with distilled water, vortexed and then heated in a water bath at 95 °C for 60 minutes.
- After cooling to room temperature, 1 ml of distilled water and 5 ml of n-butanol were added followed by vigorous shaking. After centrifugation at 4000 rpm for 10 minutes, the absorbance of the organic layer was read at 532 nm against blank containing 0.1 ml distilled water instead of the sample and treated exactly like the sample.
The accuracy of readings in TBARS assay solely depends upon freshness of sample, as you have analyzed the same sample but at a different time, therefore, you have got different readings..
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