In enhanced chemiluminescence in western blots, will the horseradish peroxidase eventually get used up? I am learning about enhanced chemiluminescence in Western blots. I have read online,-Main%20article%3A%20Chemiluminescence&text=Horseradish%20peroxidase%20catalyses%20the%20oxidation,intensity%20light%20at%20428%20nm.&text=The%20enhancement%20of%20light%20emission%20is%20called%20enhanced%20chemiluminescence%20\(ECL\).) that in enhanced chemiluminescence that horseradish peroxidase (HRP) catalyses the oxidation of luminol to 3-aminophthalate, with this reaction resulting in the emission of light. I am using HRP-conjugated secondary antibodies on my Western blots, and applying the ECL reagent to the membrane to detect the signal. I was wondering, in this chemical reaction, if the ECL reagent is left on the membrane long enough, will eventually all the HRP get used up (especially if you reapply the ECL reagent to the membrane)?

The HRP is an enzyme so it isn't a component of the reaction. By definition, enzymes only catalyze reactions but aren't an actual component of the reaction. As such they don't get used up during the reaction and are able to continually catalyze molecules until they run out of luminol in this case. The only thing to be concerned about is the membrane drying out and denaturing the HRP.

An anecdotal story, but I remember an intern dropped an extremely diluted microliter (~1/200k) of HRP conjugate into a liter bottle of TMB (A liquid that turns blue after exposure to HRP) and the entire liter bottle turned deep blue overnight. So a tiny amount of enzyme definitely didn't get used up despite a massive molar excess.