QuantiChrom Hyaluronidase Inhibitor Screening Assay Kit.

How do I calculate the concentration of test compound in the reaction?

The final concentration of the test compound in the 100 uL reaction is 5x lower than the 20 uL of test compound. So if 20 uL of 100 uM test compound was added to the well, the final concentration of test compound in the 100 uL enzymatic reaction would be 20 uM. The final concentration after adding 160 uL Stop Reagent is irrelevant because the reaction is stopped after its addition.

My percent inhibition readout is greater than 100%?

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.

My percent inhibition readout is negative?

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’s activity rather than inhibit it.

I am using a different species or a different brand of hyaluronidase, how do I determine the optimal hyaluronidase concentration for inhibitor screening?

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 uL of stock Hyaluronidase in Enzyme Buffer.
2. Transfer 40 uL of each Hyaluronidase dilution into separate wells of a clear, flat bottom 96-well plate
3. Transfer 40 uL 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 uL Substrate and 35 uL of Assay Buffer. Add 40 uL Working Reagent to sample wells and the NEC. Add 40 uL 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 uL 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.


For more detailed product information and questions, please feel free to Contact Us. Or for more general information regarding our assays, please refer to our General Questions.

RT