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

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3e+42e+41e+47.0e+38642- Dose-ResponseData legend Generated with Quest Graph™ MMP-3 (ng/well) RFU Hover mouse to interact
Dose response of MMP-3 enzyme activity was measured with Amplite™ MMP-3 Activity Assay Kit using a NOVOStar microplate reader (BMG Labtech).
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100 Tests 13512 $295


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Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
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Overview

Ex/Em (nm)498/520
SolventDMSO
Storage Freeze (<-15 °C)
Minimize light exposure
InstrumentsFluorescence microplate reader
Category Enzyme Detection
Peptidases and Proteases
Related Microplate Readers
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.




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Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.
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.

Key parameters
Instrument:Fluorescence microplate reader
Excitation:490 nm
Emission:525 nm
Cutoff:515 nm
Recommended plate:Solid black
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.

SC SC ... ...
IC IC    
VC VC    
TC TC    
TS TS    
... ...    
... ...    
       

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

Well Volume Reagent
SC 50 µL Assay Buffer (Component C)
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 assay buffer and test compound
TS 50 µL MMP-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.
Example data analysis and figures

The reading (RFU) obtained from the blank standard well is used as a negative control. Subtract this value from the other standards' readings to obtain the base-line corrected values. Then, plot the standards' readings to obtain a standard curve and equation. This equation can be used to calculate MMP-3 samples. We recommend using the Online Linear Regression Calculator which can be found at:

https://www.aatbio.com/tools/linear-logarithmic-semi-log-regression-online-calculator/

Figure 1. Dose response of MMP-3 enzyme activity was measured with Amplite™ MMP-3 Activity Assay Kit using a NOVOStar microplate reader (BMG Labtech).

Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





References & Citations

Zinc ions regulate opening of tight junction favouring efflux of macromolecules via the GSK3β/snail-mediated pathway
Authors: Ruyue Xiao, Lan Yuan, Weijiang He, Xiaoda Yang
Journal: Metallomics (2018)

Connexin 43 Upregulation by Dioscin Inhibits Melanoma Progression via Suppressing Malignancy and Inducing M1 Polarization
Authors: Yu Kou, Liyan Ji, Haojia Wang, Wensheng Wang, Hongming Zheng, Juan Zou, Linxin Liu, Xiaoxiao Qi, Zhongqiu Liu, Biaoyan Du
Journal: International Journal of Cancer (2017)

DACT2, an epigenetic stimulator, exerts dual efficacy for colorectal cancer prevention and treatment
Authors: Linlin Lu, Ying Wang, Rilan Ou, Qian Feng, Liyan Ji, Hongming Zheng, Yue Guo, Xiaoxiao Qi, Ah-Ng Tony Kong, Zhongqiu Liu
Journal: Pharmacological research (2017)

Probing Cell Adhesion Profiles with a Microscale Adhesive Choice Assay
Authors: Harsha Kittur, Andy Tay, Avery Hua, Min Yu, Dino Di Carlo
Journal: Biophysical Journal (2017): 1858--1867

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

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

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

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

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

EphA2 induces metastatic growth regulating amoeboid motility and clonogenic potential in prostate carcinoma cells
Authors: Maria Letizia Taddei, Matteo Parri, Adriano Angelucci, Francesca Bianchini, Chiara Marconi, Elisa Giannoni, Giovanni Raugei, Mauro Bologna, Lido Calorini, Paola Chiarugi
Journal: Molecular Cancer Research (2011): 149--160






Additional Documents

 
Safety Data Sheet (SDS)


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