Amplite® MMP-3 Activity Assay Kit *Green Fluorescence*
Price | |
Catalog Number | |
Unit Size | |
Quantity |
Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
Excitation (nm) | 494 |
Emission (nm) | 515 |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
Overview | SDSProtocol |
Excitation (nm) 494 | Emission (nm) 515 |
Platform
Fluorescence microplate reader
Excitation | 490 nm |
Emission | 525 nm |
Cutoff | 515 nm |
Recommended plate | Solid black |
Components
Example protocol
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 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.
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 |
-
Prepare MMP-3 containing biological samples as desired.
-
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. -
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.
Images
Citations
Authors: Covarrubias, Claudia and Cammisotto, Philippe G and Shamout, Samer and Campeau, Lysanne
Journal: Metabolites (2023): 723
Authors: Xiao, Ruyue and Yuan, Lan and He, Weijiang and Yang, Xiaoda
Journal: Metallomics (2018)
Authors: Kittur, Harsha and Tay, Andy and Hua, Avery and Yu, Min and Di Carlo, Dino
Journal: Biophysical Journal (2017): 1858--1867
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: 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: 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)
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
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
Authors: Przybyla, Laralynne M and Theunissen, Thorold W and Jaenisch, Rudolf and Voldman, Joel
Journal: Stem Cells (2013): 1097--1106
Authors: Delic, S and Lottmann, N and Jetschke, K and Reifenberger, G and Riemenschneider, Markus J
Journal: Neuropathology and applied neurobiology (2012): 201--212
Application notes
Acetylcholinesterase Inhibitory Activity of Pigment Echinochrome A
Ameliorative Effect of Novel Vitamin Formula with Herbal Extracts on Scopolamine-Induced Alzheimer's Disease
An Increase in Plasma Homovanillic Acid with Cocoa Extract Consumption Is Associated with the Alleviation of Depressive Symptoms in Overweight or Obese Adults
Attenuation of lysyl oxidase and collagen gene expression in keratoconus patient corneal epithelium corresponds to disease severity