MMP Activity Assay Kit ab112146 is designed to check the general activity of an MMP enzyme and to screen MMP inhibitors.
MMP Activity Assay Kit (Fluorometric - Green) designed for success:
- High sensitivity; the probe is much more sensitive than an EDANS/Dabcyl FRET substrate
- Easy to use and fast protocol with no wash step required.
- Can be adapted to automation without a separation step.
Fluorescent
Purified protein, Cell culture supernatant
60m
Select an associated product type
MMP Activity Assay Kit ab112146 is designed to check the general activity of an MMP enzyme and to screen MMP inhibitors.
MMP Activity Assay Kit (Fluorometric - Green) designed for success:
- High sensitivity; the probe is much more sensitive than an EDANS/Dabcyl FRET substrate
- Easy to use and fast protocol with no wash step required.
- Can be adapted to automation without a separation step.
Fluorescent
Purified protein, Cell culture supernatant
60m
Microplate reader
Blue Ice
-20°C
-20°C
-20°C
MMP Activity Assay Kit ab112146 is designed to check the general activity of an MMP enzyme and to screen MMP inhibitors.
The MMP assay protocol uses a fluorescence resonance energy transfer (FRET) peptide as a generic MMP activity indicator. In the intact FRET peptide, the fluorescence of one part is quenched by another. After cleavage into two separate fragments by MMPs, the fluorescence is recovered.
With excellent fluorescence quantum yield and longer wavelength, the probe is much more sensitive than an EDANS/Dabcyl FRET substrate. The probe signal can be easily read by a fluorescence microplate reader at Ex/Em = 490/525 nm.
MMP activity assay protocol summary:
- activate pro-MMPs with APMA for between 20 min and 24 hrs (depending on which MMP is of interest)
- add samples to wells
- add MMP Green substrate
- analyze with a fluorescent microplate reader, either after 30-60 mins for end-point reading, or every 5 min for 30-60 min for kinetic reading
The kit measures total MMP activity. It does not give an individual read-out for each MMP. The substrate peptide is a sequence that is recognized by all MMPs. A specific inihibitor would need to be included to determine the activity of a specific MMP enzyme.
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This supplementary information is collated from multiple sources and compiled automatically.
Matrix Metalloproteinases (MMPs) are a group of enzymes that break down extracellular matrix components. These are sometimes called matrixins. MMPs have a mass typically ranging from 19 to 120 kDa depending on the specific MMP enzyme. They are expressed in various tissues throughout the body including the skin lung liver and in the context of cancer cells. MMP activity is important for normal physiological processes like tissue remodeling and wound healing. Researchers frequently use MMP activity assay kits to measure the activity levels of these enzymes in biological samples for both research and clinical diagnostics.
MMPs play significant roles in tissue remodeling and cell signaling. MMPs are not usually found as part of a complex but individual enzymes can be regulated through inhibitor proteins such as TIMPs (tissue inhibitors of metalloproteinases) which bind to MMPs and modulate their enzymatic activity. This regulation is essential to maintain tissue homeostasis and any imbalance may result in pathological conditions. MMPs influence processes such as morphogenesis angiogenesis and metastasis highlighting their importance in developmental biology and cancer research.
MMPs are integral to processes involving the breakdown of the extracellular matrix. Notably MMPs are central components of the degradative pathways facilitating cell migration during immune responses and metastasis. MMPs interact closely with protein inhibitors like TIMPs and these interactions play key roles in the balance of proteolytic activity. Additionally MMPs relate to other proteins like integrins and cytokines linking them to pathways such as the TGF-beta signaling pathway and the Wnt signaling pathway.
MMPs have been implicated in conditions such as cancer and arthritis. In cancer abnormal MMP activity can elevate tumor invasion and metastasis where MMPs collaborate with proteins like E-cadherin and VEGF modulating the tumor microenvironment. In arthritis MMPs contribute to the degradation of cartilage intensifying inflammatory responses and tissue damage. Understanding MMP function and its inhibition offers therapeutic potential for managing these diseases.
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Khajah et al investigates endocrine resistant breast cancer cells in response to changes in extracellular pH. MMP activity of pII cells was determined using ab112146. Cells were cultured in a gassed or ungasssed incubator for 1 hour and treated with EGF at 50ng/ml for 30 minutes. MMP activity was then determined using fluorogenic substrate. Fluorescence was measured for excitation/emission of 490/525 nm.
The tests show the MMP activities in different matrices. 25 uL (0.31 ug) APMA-activated MMPs were spiked into 25 ul (6.25e4 cells) of cell lysate. Cells (1e6-5e7 cells) were sonicated in 0.3 mL RIPA buffer with protease inhibitors. Protein amount was determined by BCA method.
Fluorescence shown after subtraction of vehicle control (duplicates, +/- SD).
Purified MMPs used:
MMP1 – Native human MMP1 protein ab134442
MMP2 – ab125181
MMP3 – Recombinant human MMP3 protein (Active) ab96555
MMP8 – Native Human MMP8 protein ab168050
MMP9 – Native human MMP9 protein (Proenzyme, monomer) ab157344
The tests show the MMP activities in different matrices. 25 uL (0.31 ug) APMA-activated MMPs were spiked into 25 ul (6.25e4 cells) of cell lysate. Cells (1e6-5e7 cells) were sonicated in 0.3 mL RIPA buffer with protease inhibitors. Protein amount was determined by BCA method. Fluorescence shown after subtraction of vehicle control (duplicates, +/- SD).
Purified MMPs used:
MMP1 – Native human MMP1 protein ab134442
MMP2 – ab125181
MMP3 – Recombinant human MMP3 protein (Active) ab96555
MMP8 – Native Human MMP8 protein ab168050
MMP9 – Native human MMP9 protein (Proenzyme, monomer) ab157344
The tests show the MMP activities in different matrices. 25 uL (0.31 ug) APMA-activated MMPs were spiked into 25 ul (0.56 mg) of mouse liver tissue lysate. Frozen tissue was homogenised in 1.2 ml RIPA buffer with protease inhibitors with 1 mm glass beads in a bead beater. Protein was determined by BCA method.
Fluorescence shown after subtraction of vehicle control (duplicates, +/- SD).
Purified MMPs used:
MMP1 – Native human MMP1 protein ab134442
MMP2 – ab125181
MMP3 – Recombinant human MMP3 protein (Active) ab96555
MMP8 – Native Human MMP8 protein ab168050
MMP9 – Native human MMP9 protein (Proenzyme, monomer) ab157344
The tests show the MMP activities in different matrices. 25 uL (0.31 ug) APMA-activated MMPs were spiked into 25 ul (0.21 mg) of mouse leg muscle tissue lysate. Frozen tissue was homogenised in 1.2 ml RIPA buffer with protease inhibitors with 1 mm glass beads in a bead beater. Protein was determined by BCA method. Fluorescence shown after subtraction of vehicle control (duplicates, +/- SD).
Purified MMPs used:
MMP1 – Native human MMP1 protein ab134442
MMP2 – ab125181
MMP3 – Recombinant human MMP3 protein (Active) ab96555
MMP8 – Native Human MMP8 protein ab168050
MMP9 – Native human MMP9 protein (Proenzyme, monomer) ab157344
The tests show the MMP activities in different matrices. 25 uL (0.31 ug) APMA-activated MMPs were spiked into 25 ul (0.17 mg) of mouse heart tissue lysate. Frozen tissue was homogenised in 1.2 ml RIPA buffer with protease inhibitors with 1 mm glass beads in a bead beater. Protein was determined by BCA method. Fluorescence shown after subtraction of vehicle control (duplicates, +/- SD).
Purified MMPs used:
MMP1 – Native human MMP1 protein ab134442
MMP2 – ab125181
MMP3 – Recombinant human MMP3 protein (Active) ab96555
MMP8 – Native Human MMP8 protein ab168050
MMP9 – Native human MMP9 protein (Proenzyme, monomer) ab157344
The tests show the MMP activities in different matrices. 25 uL (0.31 ug) APMA-activated MMPs were spiked into 25 ul (0.80 mg) of mouse brain tissue lysate. Frozen tissue was homogenised in 1.2 ml RIPA buffer with protease inhibitors with 1 mm glass beads in a bead beater. Protein was determined by BCA method. Fluorescence shown after subtraction of vehicle control (duplicates, +/- SD).
Purified MMPs used:
MMP1 – Native human MMP1 protein ab134442
MMP2 – ab125181
MMP3 – Recombinant human MMP3 protein (Active) ab96555
MMP8 – Native Human MMP8 protein ab168050
MMP9 – Native human MMP9 protein (Proenzyme, monomer) ab157344
Detection of activity of MMPs using ab112146.
APMA-activated purified MMPs (30 ng each) were mixed with Green substrate. The fluorescence signal was monitored after 1 hour by using a microplate reader at Ex/Em = 490/525 nm. The reading from all wells was subtracted with the reading from substrate control. Although different MMPs showed different cleavage rate on this MMP substrate, the MMP Green substrate can detect the activity of sub-nanogram of all MMPs (n=3).
NOTE: distinct purified MMP enzymes were used in this test. When using cell extracts, the kit will only detect a general MMP activity and it will not differentiate between the different MMPs.
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