I am currently attempting to run a TBARS assay on algal samples under different stress conditions. I have been following the Hodges et. al, 1999 protocol from their paper, Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. I am using a spectrometer to measure the MDA equivalents but I am having difficulties getting the end quantitative values for all absorbance readings. When i perform a technical replicate on the same cuvette my absorbance values changes quite drastically. They usually increase but some do decrease. Is this reaction light sensitive? Am i detecting other compounds which increase in response to the spectrometer? I don't know how to get around this. Thanks in advance
Hi
I tried the same protocole on plant cells and it worked well..
If you have some small bubbles in your cuvettes, try to get ride of them before reading absorbance.
Good luck.
Mohammad
I have centrifuged my samples after boiling with thiobarbituric acid. This still doesn't seem to resolve the issue.
I can't remember seeing any bubbles but will check on the next run
The differences can range from 0.4 to 0.02 depending which wave length I am reading.
Ok. Do you measure the absorbance of the samples after they have cooled down?
Three steps necessary to ensure stable optical density. 1. Transparent reaction mixture (no turbidity). 2. The reaction mixture should be at room temperature before getting absorbance. 3. Some time needs calibration of spectrometer.
I hope this would help.
Ah okay thanks I think it might be that they have not fully cooled down yet. I will give this a try thank you