I am performing enzymatic inhibition assay by using flouregenic peptide containg AMC modification. I am trying to calculate percentage inhibition. But I don't know what are different controls needed for that. I am currently using (inihibitor +substrate) as sample control and calculating percentage inhibition by using formula

((Assay control- (Sample-Sample control))/Assay control)*100

Assay control contains substrate +enzyme.

But I am unable to form its IC 50 . Its looking like I am using working control. If anyone has worked on assays to find IC 50 kindly help me by providing details how to calculate and controls needed for that. Substrate +test molecule used as control because I am also getting some reading in that and subtracting it form sample.

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