I used amplex red kit for estimation of hydrogen peroxide content in Arabidopsis leaves extracted with k-phosphate buffer pH 6.4. The problem is when I make sample control reaction (i.e. extract + amplex red WITHOUT HRP) it gives same absorbance/fluorescence as extract + amplex red WITH HRP.
This means the endogenous peroxidase in the extract is enough to oxidize amplex red in presence of hydrogen peroxide. This also means for samples with same hydrogen peroxide content but different peroxidase activity will give different values for hydrogen peroxide.
this makes me skeptic about the results. I know this method is widely used ... can anyone tell if I am wrong?
If your goal is to measure the H2O2 content of samples, not the peroxidase content, then it will be important to make sure that the peroxidase concentration in the assay is not a limiting factor, no matter what the H2O2 concentration.Therefore, you will have to be sure to add enough peroxidase so that you can get a good standard curve for H2O2 in the desired range, thereby eliminating the sample peroxidase content as a factor.
You might also be interested in measuring the peroxidase content of the samples rather than the H2O2 content. In that case, you should add sufficient H2O2 so that you can get a good standard curve of peroxidase activity regardless of the H2O2 content of the samples.
Hello Walid,
in the attachment you will find a protocol for measuring hydrogen peroxide content in A. thaliana leaf discs using Amplex Red. Maybe this protocol can give you some insights or clues where your protocol might go wrong?
Regards, Christian
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