I homogenized murine lungs in 0.5% w/v HDAB in 50mM sodium phosphate buffer to perform a MPO activity assay. The protocol I do have instructs that I should use 20ul of my samples with 160ul TMB and 20ul H2O2 in 80mM sodium phosphate buffer and wait for 5' to see colour change.
However, it does not specify what the concentration of H2O2 in sodium phosphate buffer should be. Anyone who has some experience with this technique?
A 30% stock concentration of H202 is about 8800 mM. A 3% stabilized solution of H2O2 is 880 mM. I have worked several peroxidase assays, and they require 1 mM H2O2 (0.003%) or less. A few Myeloperoxidase (MPO) assays I have seen require 0.01 and 0.1 mM H2O2. You really do not need a lot of H2O2, as it may compromise your other assay components. Maximum I would use for H2O2 assays in general is 1 mM. For MPO, max may be 0.1 mM- you can also follow the progress and see if you get substrate depletion.
Hi everyone,
thanks for your replies. I tried 0.003%, 0.001% and o.0005% H2O2 in the sodium phosphate buffer and it worked fine in all three cases. However, as many kit protocols recommended concentrations around 0.001-0.002%, I decided to use 0.001% in future experiments, as I think H2O2 is anyway in excess at these concentrations.
Take care in not including NaN2 as preservative in any of buffers, that might prevent peroxidase activity.
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