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MMP-3 Green™ substrate solution
The matrix metalloproteinases (MMPs) constitute a family of zinc-dependent endopeptidases that function within the extracellular matrix. These enzymes are responsible for the breakdown of connective tissues and are important in bone remodeling, the menstrual cycle and repair of tissue damage. While the exact contribution of MMPs to certain pathological processes is difficult to assess, MMPs appear to play a key role in the development of arthritis as well as in the invasion and metastasis of cancer. MMPs tend to have multiple substrates, with most family members having the ability to degrade different types of collagen along with elastin, gelatin and fibronectin. It is quite difficult to find a substrate that is selective to a single MMP enzyme. This FRET substrate is designed to have a relative high selectivity to MMP-3. It is used to monitor the MMP-3 activity. It can also be used to screening MMP-3 inhibitors when a purified MMP-3 enzyme is used. This FRET substrate is based on our TF2/TQ2 FRET pair. Upon MMP-3 hydrolysis the fluorescence of MMP-3 Green™ FRET peptide substrate is increased since the TF2/TQ2 FRET pair is separated. The fluorescence increase is proportional to the MMP-3 enzyme activities.
Example protocol
AT A GLANCE
- Add appropriate controls, or test samples (50 µL)
- Pre-incubate for 10 - 15 minutes
- Add MMP-3 Green™ substrate working solution (50 µL)
- Skip incubation for kinetic reading or incubate 30 to 60 minutes for end point reading
- Monitor fluorescence intensity at Ex/Em = 490/525 nm
PREPARATION OF WORKING SOLUTION
SAMPLE EXPERIMENTAL PROTOCOL
- Prepare MMP-3 containing biological samples as desired.
- Activate pro-MMP-3 as per protocol. Note: 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.
- 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.
- 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.
- 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.
- 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.
| SC | SC | ... | ... |
| IC | IC | ||
| VC | VC | ||
| TC | TC | ||
| TS | TS | ||
| ... | ... | ||
| ... | ... | ||
| Well | Volume | Reagent |
| SC | 50 µL | Buffer of your choice |
| IC | 50 µL | MMP-3 dilution and known MMP-3 inhibitor |
| VC | 50 µL | MMP-3 dilution and vehicle used to deliver test compound |
| TC | 50 µL | MMP-3 containing buffer and test compound |
| TS | 50 µL | MMP-3 dilution with test compound |
Safety data sheet