MMP Green™ substrate

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Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
International	See distributors
Bulk request	Inquire
Custom size	Inquire
Shipping	Standard overnight for United States, inquire for international

Physical properties

Molecular weight	~2000
Solvent	DMSO

Spectral properties

Excitation (nm)	494
Emission (nm)	515

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12352200

Alternative formats

Overview SDS Protocol

Platform

Fluorescence microplate reader

Excitation	490 nm
Emission	525 nm
Cutoff	515 nm
Recommended plate	Solid black

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

Important Thaw the reagents includig the substrate at room temperature before starting the experiment. Prepare MMPs containing biological samples as desired.

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.

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

Open in Advanced Spectrum Viewer

Spectral properties

Excitation (nm)	494
Emission (nm)	515

Product Family

Name	Excitation (nm)	Emission (nm)
MMP Red™ substrate	545	572

Images

Figure 1. The internally quenched FRET peptide substrate is digested by a protease to generate the highly fluorescent peptide fragment. The fluorescence increase is proportional to the protease activity.

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

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)

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)

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)

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

Inhibition of BET bromodomain attenuates angiotensin II induced abdominal aortic aneurysm in ApoE-/- mice
Authors: Duan, Qiong and Mao, Xiaoxiao and Liao, Chaonan and Zhou, Haoyang and Sun, Zelin and Deng, Xu and Hu, Qiuning and Qi, Jun and Zhang, Guogang and Huang, He and others, undefined
Journal: International Journal of Cardiology (2016): 428--432

Tissue inhibitor of metalloproteinase 1 influences vascular adaptations to chronic alterations in blood flow
Authors: M, undefined and el, Erin R and Uchida, Cass and ra , undefined and Nwadozi, Emmanuel and Makki, Armin and Haas, Tara L
Journal: Journal of Cellular Physiology (2016)

Matrix Remodeling Maintains Embryonic Stem Cell Self-Renewal by Activating Stat3
Authors: Przybyla, Laralynne M and Theunissen, Thorold W and Jaenisch, Rudolf and Voldman, Joel
Journal: Stem Cells (2013): 1097--1106

β-adrenoceptors are upregulated in human melanoma and their activation releases pro-tumorigenic cytokines and metalloproteases in melanoma cell lines
Authors: Moretti, Silvia and Massi, Daniela and Farini, Valentina and Baroni, Gianna and Parri, Matteo and Innocenti, Stefania and Cecchi, Roberto and Chiarugi, Paola
Journal: Laboratory Investigation (2013): 279--290

Identification and functional validation of CDH11, PCSK6 and SH3GL3 as novel glioma invasion-associated candidate genes
Authors: Delic, S and Lottmann, N and Jetschke, K and Reifenberger, G and Riemenschneider, Markus J
Journal: Neuropathology and applied neurobiology (2012): 201--212