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Amplite® MMP-3 Activity Assay Kit *Green Fluorescence*

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

Protocol summary

  1. Add appropriate controls, or test samples (50 µL)
  2. Pre-incubate for 10 - 15 minutes
  3. Add MMP-3 Green™ substrate working solution (50 µL)
  4. Skip incubation for kinetic reading or incubate 30 to 60 minutes for end point reading
  5. Monitor fluorescence intensity at Ex/Em = 490/525 nm

Important notes
Thaw all the kit components 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 (Component A) into 5 mL of Assay Buffer (Component C) to make a total volume of 5.05 mL.

2. MMP-3 dilution:
Dilute MMP-3 to an appropriate concentration in Assay Buffer (Component C) 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

Table 1. Layout of the appropriate controls (as desired) and test samples in a 96-well microplate. SC= Substrate Control, IC= Inhibitor Control, VC=Vehicle Control, TC= Test Compound Control, TS=Test Samples.

SCSC......
ICIC  
VCVC  
TCTC  
TSTS  
......  
......  
    

Table 2. Reagent composition for each well. Note: Some strongly fluorescent test compounds may result in false-positive results.

WellVolumeReagent
SC50 µLAssay Buffer (Component C)
IC50 µLMMP-3 dilution and known MMP-3 inhibitor
VC50 µLMMP-3 dilution and vehicle used to deliver test compound
TC50 µLMMP-3 containing assay buffer and test compound
TS50 µLMMP-3 dilution with test compound
  1. Prepare MMP-3 containing biological samples as desired.

  2. To activate pro-MMP-3, first dilute 1M APMA (Component B) with Assay Buffer (Component C) at 1:500 to get a 2 mM APMA working solution (2X). Note: APMA belongs to organic mercury. Handle with care! Dispose it according to local regulations.

    Next, 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. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

Citations

View all 11 citations: Citation Explorer
Decrease in the Ratio proBDNF/BDNF in the Urine of Aging Female Patients with OAB
Authors: Covarrubias, Claudia and Cammisotto, Philippe G and Shamout, Samer and Campeau, Lysanne
Journal: Metabolites (2023): 723
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)
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
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)
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)
Page updated on October 14, 2024

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Catalog Number13512
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Spectral properties

Excitation (nm)

494

Emission (nm)

515

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

Platform

Fluorescence microplate reader

Excitation490 nm
Emission525 nm
Cutoff515 nm
Recommended plateSolid black

Components

Dose response of MMP-3 enzyme activity was measured with Amplite® MMP-3 Activity Assay Kit using a NOVOStar microplate reader (BMG Labtech).
Dose response of MMP-3 enzyme activity was measured with Amplite® MMP-3 Activity Assay Kit using a NOVOStar microplate reader (BMG Labtech).
Dose response of MMP-3 enzyme activity was measured with Amplite® MMP-3 Activity Assay Kit using a NOVOStar microplate reader (BMG Labtech).