How much inhibitor should I add to my enzyme reaction, HELP PLEASE.

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abd265
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How much inhibitor should I add to my enzyme reaction, HELP PLEASE.

Hello everybody,
I am trying to test different  inhibitors on one enzyme and the problem is I cant find any papers that tell exactly the amount of inhibitor i should add; This is what i found in one of these many papers talking about the reaction:

'' Figure 2. Residual Enzyme activity after treatment with 50 nM inhibitor compounds in vitro. Recombinant Enzyme was preincubated with eight inhibitors for 5 min and then incubated with 3 nmol of substrate for 30 min.
Enzyme activity was measured by colorimetric determination of inorganic PO4 3– levels and is shown relative to the
activity of uninhibited enzyme (control) standard error (SE) of triplicate experiments.

My question is: Should i prepare different inhibitor solutions with 50nM of concentration then take an amount of it? if yes how much volume should i take? (total volume of Enzyme+substrate+inhibitor = 25 µl);
Or should I prepare slutions with any concentrations then add a specific amount of the inhibitor to make it 50nM in the final volume? if yes what is the final vomule to be considered: enzyme+inhibitor only, before adding the substrate or the volume of the enzyme+substrate+inhibitor?

Thank you a lot for you help
Have a nice day;