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MMP Green™ substrate
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 monitor the general activity of a MMP enzyme. It can also be used to screening MMP inhibitors when a purified MMP enzyme is used. This FRET substrate is based on our TF2/TQ2 FRET pair. Upon MMP hydrolysis the fluorescence of MMP Green™ FRET peptide substrate is increased since the TF2/TQ2 FRET pair is separated. The fluorescence increase is proportional to the MMP enzyme activities.
Example protocol
AT A GLANCE
Protocol summary
- Add appropriate controls or test samples with or without APMA (50 µL)
- Pre-incubate for 10 - 15 minutes
- Add MMP Green™ substrate working solution (50 µL)
- Skip incubation step for kinetic reading or incubate for 30 to 60 minutes for end point reading
- Monitor fluorescence intensity at Ex/Em = 490/525 nm
PREPARATION OF STOCK SOLUTIONS
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.
Note Store stock solution in single used aliquots and protect from light.
MMP Green™ Substrate stock solution
For Cat# 13520, prepare 1- 10 mg/mL stock solution by adding appropriate amount of DMSO to MMP Green™ substrate vial.Note Store stock solution in single used aliquots and protect from light.
PREPARATION OF WORKING SOLUTION
MMP Green™ Substrate working solution
For Cat# 13520, prepare 20-100 µg/mL MMP Green™ Substrate working solution in buffer of your choice. For Cat# 13519, Add 50 µL of MMP Green™ solution (vial provided) into 5 mL of buffer of your choice.SAMPLE EXPERIMENTAL PROTOCOL
Table 1.Protocols for MMP activation.
| MMPs | Activated by treating with |
| MMP-1 (collagenase) | 1 mM APMA (diluted component C) at 37 °C for 3 hr |
| MMP-2 (gelatinase) | 1 mM APMA (diluted component C) at 37 °C for 1 hr |
| MMP-3 (stromelysin) | 1 mM APMA (diluted component C) at 37 °C for 24 hr |
| MMP-7 (matrilysin, PUMP-1) | 1 mM APMA (diluted component C) at 37 °C for 20 min - 1 hr |
| MMP-8 (neutrophil collagenase) | 1 mM APMA (diluted component C) at 37 °C for 1 hr |
| MMP-9 (92 kDa gelatinase) | 1 mM APMA (diluted component C) at 37 °C for 2 h |
| MMP-10 (stromelysin-2) | 1 mM APMA (diluted component C) at 37 °C for 24 hr |
| MMP-11 (stromelysin-3) | Already in active form. No APMA treatment is necessary |
| MMP-12 (macrophage elastase) | 1 mM APMA (diluted component C) at 37 °C for 2 hr |
| MMP-13 (collagenase-3) | 1 mM APMA (diluted component C) at 37°C for 40 min |
| MMP-14 | 1 mM APMA (diluted component C) at 37°C for 2 - 3 hr |
- Prepare MMPs containing biological samples as desired.
- To activate pro-MMPs, add 2 mM APMA working solution (2X) to the plate.
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 1 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. - Prepare controls and test samples (TS) with 50 µL per well. 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 MMPs inhibitors.
- 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.
- 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.
Spectrum
Product family
| Name | Excitation (nm) | Emission (nm) |
| MMP Red™ substrate | 545 | 572 |
Citations
View all 11 citations: Citation Explorer
Octenidine-based hydrogel shows anti-inflammatory and protease-inhibitory capacities in wounded human skin
Authors: Seiser, Saskia and Janker, Lukas and Zila, Nina and Mildner, Michael and Rakita, Ana and Matiasek, Johannes and Bileck, Andrea and Gerner, Christopher and Paulitschke, Verena and Elbe-B{\"u}rger, Adelheid
Journal: Scientific Reports (2021): 1--13
Authors: Seiser, Saskia and Janker, Lukas and Zila, Nina and Mildner, Michael and Rakita, Ana and Matiasek, Johannes and Bileck, Andrea and Gerner, Christopher and Paulitschke, Verena and Elbe-B{\"u}rger, Adelheid
Journal: Scientific Reports (2021): 1--13
Zinc ions regulate opening of tight junction favouring efflux of macromolecules via the GSK3β/snail-mediated pathway
Authors: Xiao, Ruyue and Yuan, Lan and He, Weijiang and Yang, Xiaoda
Journal: Metallomics (2018)
Authors: Xiao, Ruyue and Yuan, Lan and He, Weijiang and Yang, Xiaoda
Journal: Metallomics (2018)
Probing Cell Adhesion Profiles with a Microscale Adhesive Choice Assay
Authors: Kittur, Harsha and Tay, Andy and Hua, Avery and Yu, Min and Di Carlo, Dino
Journal: Biophysical Journal (2017): 1858--1867
Authors: Kittur, Harsha and Tay, Andy and Hua, Avery and Yu, Min and Di Carlo, Dino
Journal: Biophysical Journal (2017): 1858--1867
DACT2, an epigenetic stimulator, exerts dual efficacy for colorectal cancer prevention and treatment
Authors: Lu, Linlin and Wang, Ying and Ou, Rilan and Feng, Qian and Ji, Liyan and Zheng, Hongming and Guo, Yue and Qi, Xiaoxiao and Kong, Ah-Ng Tony and Liu, Zhongqiu
Journal: Pharmacological research (2017)
Authors: Lu, Linlin and Wang, Ying and Ou, Rilan and Feng, Qian and Ji, Liyan and Zheng, Hongming and Guo, Yue and Qi, Xiaoxiao and Kong, Ah-Ng Tony and Liu, Zhongqiu
Journal: Pharmacological research (2017)
Connexin 43 Upregulation by Dioscin Inhibits Melanoma Progression via Suppressing Malignancy and Inducing M1 Polarization
Authors: Kou, Yu and Ji, Liyan and Wang, Haojia and Wang, Wensheng and Zheng, Hongming and Zou, Juan and Liu, Linxin and Qi, Xiaoxiao and Liu, Zhongqiu and Du, Biaoyan and others, undefined
Journal: International Journal of Cancer (2017)
Authors: Kou, Yu and Ji, Liyan and Wang, Haojia and Wang, Wensheng and Zheng, Hongming and Zou, Juan and Liu, Linxin and Qi, Xiaoxiao and Liu, Zhongqiu and Du, Biaoyan and others, undefined
Journal: International Journal of Cancer (2017)
Page updated on November 21, 2024
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