Screen Quest™ Fluorimetric Neutrophil Elastase Inhibitor Screening Kit

1. Prepare the NE enzyme solution (40 μL/well).

2. Prepare the NE inhibitor serial dilution, and add 10 μL/well.

3. Incubate the NE enzyme with inhibitor for 5-10 minutes at 37 °C.

4. Add 50 µL of the NE working solution.

5. Incubate at room temperature for 10-30 minutes.

6. Monitor fluorescence intensity at Ex/Em = 360/470 nm, Cutoff = 430 nm.

Thaw all the kit components at room temperature before starting the experiment.

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

1. Add 50 µL of the NE Assay Buffer (Component D) to the vial of NE Enzyme (Component B) to make a 100 µg/mL NE Enzyme stock solution. Mix well by pipetting, aliquot, and store at -80 °C.

   Note: Must be used within 1 week of reconstitution. Avoid freeze/thaw cycles.

1. Take 5 μL of the 100 μg/mL NE Enzyme stock solution and add it to 495 μL of NE Assay Buffer (Component D) to create a 1 μg/mL NE Enzyme solution.

   Note: The concentration of the NE Enzyme solution needs to be optimized when using a different inhibitor.

1. Add 25 µL of NE Substrate (100X) (Component A) to 5 mL of NE Assay Buffer (Component D), and mix well.

   Note: Prepare the NE working solution fresh before each experiment, and protect it from light.

   Note: A 5 mL solution is sufficient for 100 tests. Adjust the amount of NE working solution proportionally to match the number of tests you need.

Table 1. Layout of NE inhibitor serial dilution and test samples in a 96-well solid black microplate. (STD = NE Inhibitor Standards (STD1-STD7, 1.25-80 µM), BL= Blank Control, TS = Test Samples.)

Table 2. Reagent composition for each well.

1. Prepare NE Inhibitor standards (STD1-7), blank controls (BL), positive control, and test samples (TS) as outlined in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.

2. Incubate the NE enzyme with inhibitor for 5-10 minutes at 37 ⁰C.

3. Add 50 µL of NE Substrate Working Solution to each well containing the NE Inhibitor standards (STD1-STD7), blank control, positive control, and test samples. For a 384-well plate, add 25 µL of GGT Working Solution to each well instead.

4. Incubate for 10-30 minutes at 37 °C, protected from light.

5. Monitor the fluorescence intensity with a fluorescence microplate reader at Ex/Em = 360/470 nm (Cutoff = 430 nm).