The final concentration of the test compound in the 100 µL reaction is 5× lower than the 20 µL of test compound. So if 20 µL of 100 µM test compound was added to the well, the final concentration of test compound in the 100 µL enzymatic reaction would be 20 µM. The final concentration after adding 160 µL Stop Reagent is irrelevant because the reaction is stopped after its addition.
Inhibition readouts slightly over 100% (e.g. 100% to 110%) are not an issue and can be assumed to just be 100% inhibition. Inhibition readouts significantly over 100% may mean there is a substance interfering with the assay.
Inhibition readouts that are slightly negative (e.g. -1% to -10%) are not an issue and can be assumed to be 0% inhibition. Inhibition readouts that are significantly negative may mean there is a substance interfering with the assay or that the test compound is acting as an activator to improve the enzyme activity rather than inhibit it.
How to Determine Optimal Hyaluronidase Concentration for Inhibitor Screening:
1. Prepare stock Hyaluronidase solution in Enzyme Buffer (start at a high concentration, you can always dilute down). In eppendorf tubes, serially dilute 50 µL of stock Hyaluronidase in Enzyme Buffer.
2. Transfer 40 µL of each Hyaluronidase dilution into separate wells of a clear, flat bottom 96-well plate
3. Transfer 40 µL of dH2O into two separate wells for the No Enzyme Control (NEC) and No Substrate Control (NSC).
4. Prepare enough Working Reagent for each well by combining 10 µL Substrate and 35 µL of Assay Buffer. Add 40 µL Working Reagent to sample wells and the NEC. Add 40 µL Assay Buffer to the NSC well.
6. Tap plate briefly to mix and incubate the plate for 20 minutes at room temperature.
7. Add 160 µL Stop Reagent to each well. Tap plate to mix briefly and thoroughly.
8. Incubate for 10 minutes at room temperature and read optical density at 600 nm.
Select a concentration of enzyme that yields an OD600nm reading about half way between the NEC and NSC OD600nm readings.
Note: the low concentrations of enzyme may produce OD600nm readings slightly higher than the NEC, this is normal.
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