Amplite® MMP-3 Activity Assay Kit *Green Fluorescence*

Protocol summary

1. Add appropriate controls, or test samples (50 µL)
2. Pre-incubate for 10 - 15 minutes
3. Add MMP-3 Green™ substrate working solution (50 µL)
4. Skip incubation for kinetic reading or incubate 30 to 60 minutes for end point reading
5. Monitor fluorescence intensity at Ex/Em = 490/525 nm

Important notes
Thaw all the kit components at room temperature before starting the experiment. Prepare MMP-3 containing biological samples as desired.

1. MMP-3 Green™ Substrate working solution:
Add 50 μL of MMP-3 Green™ Substrate (Component A) into 5 mL of Assay Buffer (Component C) to make a total volume of 5.05 mL.

2. MMP-3 dilution:
Dilute MMP-3 to an appropriate concentration in Assay Buffer (Component C) if purified MMP-3 is used. Note: MMP-3 needs to be activated before use. Avoid vigorous vortexing of the enzyme.

3. Inhibitors and compounds dilution:
Make an appropriate concentration of known MMP-3 inhibitors and test compounds dilutions as desired if screening MMP-3 inhibitors.

Table 1. Layout of the appropriate controls (as desired) and test samples in a 96-well microplate. SC= Substrate Control, IC= Inhibitor Control, VC=Vehicle Control, TC= Test Compound Control, TS=Test Samples.

Table 2. Reagent composition for each well. Note: Some strongly fluorescent test compounds may result in false-positive results.

1. Prepare MMP-3 containing biological samples as desired.

2. To activate pro-MMP-3, first dilute 1M APMA (Component B) with Assay Buffer (Component C) at 1:500 to get a 2 mM APMA working solution (2X). Note: APMA belongs to organic mercury. Handle with care! Dispose it according to local regulations.
   
   Next, incubate the MMP-3 containing-samples or purified MMP-3 with equal volume of 2 mM APMA working solution (2X) at 37 °C for 24 hours. Activate MMP-3 immediately before the experiment. Note: Keep enzyme-containing samples on ice. Avoid vigorously vortexing the enzyme. Prolonged storage of the activated enzyme will deactivate the enzyme. For enzyme activation, it is preferably activated at higher protein concentration. After activation, you may further dilute the enzyme.

3. Prepare controls and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 20 µL of reagent per well instead of 50 µL.

4. Pre-incubate the plate at a desired temperature for the enzyme reaction (e.g. 25 °C or 37 °C) for 10 - 15 minutes if you are screening MMP-3 inhibitors.
   
   
5. Add 50 µL (96-well) or 20 µL (384-well) of MMP-3 Green™ substrate working solution to the sample and control wells of the assay plate. Mix the reagents well.
   
   
6. Monitor the fluorescence intensity with a fluorescence plate reader at Ex/Em = 490/525 nm.
   
   For kinetic reading: Immediately start measuring fluorescence intensity and continuously record data every 5 minutes for 30 to 60 minutes.
   
   For end-point reading: Incubate the reaction at room temperature for 30 to 60 minutes, kept from light if possible. Mix the reagents well, and then measure the fluorescence intensity.