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MMP-3 Green™ substrate solution
Example protocol
AT A GLANCE
AT A GLANCE
Protocol Summary
- 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
Important
Thaw the solution at room temperature before starting the experiment. Prepare MMP-3 containing biological samples as desired.PREPARATION OF WORKING SOLUTION
1. MMP-3 Green™ Substrate working solution
Add 50 μL of MMP-3 Green™ Substrate Solution into 5 mL of buffer of your choice to make a total volume of 5.05 mL. Note: Tris buffer can be used for the assay.2. MMP-3 dilutions
Dilute MMP-3 to an appropriate concentration in buffer of your choice 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.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 |
Spectrum
Page updated on October 14, 2024
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