Amplite® Universal Fluorimetric MMP 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 Green™ substrate working solution (50 µL)
4. Skip incubation step for kinetic reading or incubate for 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 MMPs containing biological samples as desired.

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

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

3. Inhibitors and compounds dilution:
Make dilutions of known MMPs inhibitors and test compounds as desired if you are screening MMPs 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. Some strongly fluorescent test compounds may result in false-positive results. Make the total volume of all the controls to 50 µL for a 96-well plate or 20 µL for a 384-well plate by using Assay Buffer (Component C).

Table 3. Protocols for MMP activation.

1. Prepare MMPs containing biological samples as desired. 
   
   
2. To activate pro-MMPs, first dilute 1 M APMA (Component B) with Assay Buffer (Component C) at 1:500 to get 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 containing-samples or purified MMPs with equal volume of 2 mM APMA working solution (2X). Refer to Table 3 for incubation time. Activate MMPs 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 min if you are screening MMPs inhibitors.
   
   
5. Add 50 µL (96-well) or 20 µL (384-well) of MMP 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.