Amplex Red & HRP non- specific interaction

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Andy_Kusiak's picture
Amplex Red & HRP non- specific interaction

Hi all,

I am quite new to the biotechnology world and was wondering whether you would be able to help me out on a problem i am facing at the moment.

I am currently working on an enzymatic cholesterol assay with a fluorescent reporter (Amplex Red). This is the standard assay by where you measure the amount of fluorescence generated by the interaction of H2O2 and the Amplex red probe in the presence of HRP.

I am looking to dry these two compononts down together and test as part of a mini-stability trial. Unfortunately it seems as though i am seeing a steady increase in signal generated overtime when tested with a cholesterol free sample.  Some literature suggests that this is due to the independent interaction of HRP with the Amplex Red.

Has anyone every seen this before? and if so do you know what brings about this interaction?