Amplite® Universal Fluorimetric MMP Activity Assay Kit *Red Fluorescence*
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Unit Size | |
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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) | 545 |
Emission (nm) | 572 |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
Amplite® Universal Fluorimetric MMP Activity Assay Kit *Green Fluorescence* |
Overview | SDSProtocol |
Excitation (nm) 545 | Emission (nm) 572 |
Platform
Fluorescence microplate reader
Excitation | 540 nm |
Emission | 590 nm |
Cutoff | 570 nm |
Recommended plate | Solid black |
Components
Example protocol
AT A GLANCE
Protocol summary
- Prepare appropriate controls or test samples (50 µL)
- Pre-incubate for 10 -15 minutes
- Add MMP RedTM Substrate working solution (50 µL)
- Skip incubation for kinetic reading or incubate 30 minutes - 1 hour for end point reading
- Monitor fluorescence intensity at Ex/Em = 540/590 nm (Cutoff = 570 nm)
Important notes
Thaw all the kit Components at room temperature before starting the experiment.
PREPARATION OF WORKING SOLUTION
1. APMA working solution (2mM, 2X):
Dilute 1 M APMA (Component B) with Assay Buffer (Component C) at 1:500 to make 2 mM, 2X APMA working solution . Note: APMA belongs to organic mercury. Handle with care! Dispose it according to local regulations.
2. MMP RedTM Substrate working solution:
Add 50 µL of MMP RedTM Substrate (Component A) to 5 mL of Assay Buffer (Component C) and mix well to make MMP RedTM Substrate working solution. Note: MMP RedTM Substrate working solution is enough for one 96-well plate (100 assays).
3. MMP dilution:
Dilulte MMPs to an appropriate concentration with Assay Buffer (Component C) if purified MMP is used. Note: Pro-MMP needs to be activated before use. Avoid vigorously vortexing the enzyme.
4. Inhibitors and compounds dilutions:
Make dilutions of known MMPs inhibitors and test compounds as desired if you are screening MMPs inhibitors.
For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Protocols for pro-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 hr. |
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. |
Table 2. Layout of the samples in a solid black 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 3. Reagent composition for each well. Some strongly fluorescent test compounds may result in false-positive results.
Well | Volume | Reagent |
SC | 50 µL | Assay Buffer |
IC |
50 µL |
MMP dilution and known MMPs inhibitor |
VC | 50 µL |
MMP dilution and vehicle used to deliver test compound |
TC | 50 µL | Assay Buffer and test compound |
TS | 50 µL |
MMP dilution with test compound |
- Prepare MMPs containing biological samples as desired.
- 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 MMP 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. Note: For enzyme activation, it is preferably activated at higher protein concentration. After activation, you may further dilute the enzyme.
- Prepare Subtrate Control (SC), Inhibitor Control (IC), Vehicle Control (VC), Test Compound Control (TC) and Test Samples (TS) according to the layout provided in Table 2 and Table 3.
- Pre-incubate the plate at a desired temperature for the enzyme reaction (e.g. 25 °C or 37 °C) for 10-15 min if you are screening MMPs inhibitors.
- Add 50 µL/well (96-well plate) or 20 µL/well (384-well plate) of MMP Red™ 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 = 540/590 nm (Cutoff = 570 nm).
For kinetic reading: Immediately start measuring fluorescence intensity continuously and record data every 5 minutes for 30 minutes.
For end-point reading: Incubate the reaction at a desired temperature for 30 to 60 minutes, protected from light. Mix the reagents well, and then measure the fluorescence intensity.
Product Family
Name | Excitation (nm) | Emission (nm) |
Amplite® Universal Fluorimetric MMP Activity Assay Kit *Green Fluorescence* | 494 | 515 |
Images
Citations
Authors: Boddu, Vijay Kumar and Zamzow, Piet and Kramer, Mario Wolfgang and Merseburger, Axel S and Gorantla, Sivahari Prasad and Klinger, Matthias and Cramer, Lena and Sauer, Thorben and Gemoll, Timo and von Bubnoff, Nikolas and others,
Journal: Cell Communication and Signaling (2024): 1--14
Authors: Shieh, Jiunn-Min and Chang, Ting-Wei and Wang, Jing-He and Liang, Song-Ping and Kao, Pei-Lu and Chen, Liang-Yi and Yen, Chia-Jui and Chen, Yun-Ju and Chang, Wen-Chang and Chen, Ben-Kuen
Journal: The FASEB Journal (2023): e23206
Authors: Akentieva, Natalia and Sanina, Natalia and Gizatullin, Artur and Shkondina, Natalia and Andreeva, Anna and Shram, Stanislav and Aldoshin, Sergei
Journal: (2022)
Authors: Xiao, Ruyue and Yuan, Lan and He, Weijiang and Yang, Xiaoda
Journal: Metallomics (2018)
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,
Journal: International Journal of Cancer (2017): 1690--1703
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: 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: Kittur, Harsha and Tay, Andy and Hua, Avery and Yu, Min and Di Carlo, Dino
Journal: Biophysical Journal (2017): 1858--1867
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: 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)
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