Amplite® Universal Fluorimetric MMP Activity Assay Kit *Red Fluorescence*

Protocol summary

1. Prepare appropriate controls or test samples (50 µL)
2. Pre-incubate for 10 -15 minutes 
3. Add MMP Red^TM Substrate working solution (50 µL)
4. Skip incubation for kinetic reading or incubate 30 minutes - 1 hour for end point reading
5. Monitor fluorescence intensity at Ex/Em = 540/590 nm (Cutoff = 570 nm)

Important notes
Thaw all the kit Components at room temperature before starting the experiment. 

1. APMA working solution (2mM, 2X):
Dilute 1 M APMA (Component B) with Assay Buffer (Component C) at 1:500 to make 2 mM, 2X APMA working solution . Note: APMA belongs to organic mercury. Handle with care! Dispose it according to local regulations.

2. MMP Red^TM Substrate working solution:
Add 50 µL of MMP Red^TM Substrate (Component A) to 5 mL of Assay Buffer (Component C) and mix well to make MMP Red^TM Substrate working solution. Note: MMP Red^TM Substrate working solution is enough for one 96-well plate (100 assays).

3. MMP dilution:
Dilulte MMPs to an appropriate concentration with Assay Buffer (Component C) if purified MMP is used. Note: Pro-MMP needs to be activated before use.  Avoid vigorously vortexing the enzyme.

4. Inhibitors and compounds dilutions:
Make dilutions of known MMPs inhibitors and test compounds as desired if you are screening MMPs inhibitors.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

Table 1. Protocols for pro-MMP activation

Table 2. Layout of the samples in a solid black 96-well microplate.  SC=Substrate Control, IC=Inhibitor Control, VC=Vehicle Control, TC=Test Compound Control, TS=Test Samples. 

Table 3. Reagent composition for each well. Some strongly fluorescent test compounds may result in false-positive results.

1. Prepare MMPs containing biological samples as desired.
   
   
2. Incubate the MMP containing-samples or purified MMPs with equal volume of 2 mM APMA working solution (2X). Refer to Table 1 for incubation time. Activate MMP 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. Note: For enzyme activation, it is preferably activated at higher protein concentration. After activation, you may further dilute the enzyme.
   
   
3. Prepare Subtrate Control (SC), Inhibitor Control (IC), Vehicle Control (VC), Test Compound Control (TC) and Test Samples (TS) according to the layout provided in Table 2 and Table 3.
   
   
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/well (96-well plate) or 20 µL/well (384-well plate) of MMP Red™ 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 = 540/590 nm (Cutoff = 570 nm).
   
   For kinetic reading: Immediately start measuring fluorescence intensity continuously and record data every 5 minutes for 30 minutes.
   
   For end-point reading: Incubate the reaction at a desired temperature for 30 to 60 minutes, protected from light.  Mix the reagents well, and then measure the fluorescence intensity.