Recombinant Human MMP-14/MT1-MMP (NS0-expressed) Protein, CF
Recombinant Human MMP-14/MT1-MMP (NS0-expressed) Protein, CF Summary
Product Specifications
Ala21-Ser538, with an Arg108Pro mutation and a C-terminal 6-His tag
Analysis
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
918-MPN
| Formulation | Supplied as a 0.2 μm filtered solution in Sodium Acetate, NaCl, CaCl2, Glycerol. |
| Shipping | The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Assay Procedure
- Assay Buffer: 50 mM Tris, 0.15 M NaCl, 10 mM CaCl2, 5 µM ZnCl2, 0.05% (w/v) Brij-35, pH 7.5
- Recombinant Human MMP‑14/MT1‑MMP (rhMMP-14) (Catalog # 918-MPN)
- Recombinant Human Trypsin 3 (rhTrypsin 3) (Catalog # 3714-SE)
- AEBSF (Catalog # 5175), 100 mM stock in deionized water
- Substrate: MCA-Lys-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH2 (Catalog # ES010), 2 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Activate rhMMP-14 at 100 µg/mL with 0.1 µg/mL rhTrypsin 3 in Assay Buffer.
- Incubate at 37 °C for 1 hour.
- Stop reaction with 1 mM AEBSF and incubate for 15 minutes at room temperature.
- Dilute activated rhMMP-14 to 1 µg/mL in Assay Buffer.
- Dilute Substrate to 80 µM in Assay Buffer.
- In a plate, load 50 µL of 1 µg/mL rhMMP-14 to wells. For a Substrate Blank, load 50 µL of Assay Buffer.
- Start the reaction by adding 50 µL of 80 µM Substrate to wells.
- Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
- Calculate specific activity:
|
Specific Activity (pmol/min/µg) = |
Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU) |
| amount of enzyme (µg) |
*Adjusted for Substrate Blank
**Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).
- rhMMP-14: 0.050 µg
- Substrate: 40 µM
Reconstitution Calculator
Background: MMP-14/MT1-MMP
The first identified member of membrane type (MT) MMPs, MMP-14, also known as MT1-MMP, plays an important role in extracellular matrix (ECM) remodeling by being able to degrade type 1 collagen, activate pro-MMP-2 and process cell adhesion molecules such as CD44 and integrin alpha v (1). MMP‑14 is therefore a key enzyme in many physiological and pathological processes such as angiogenesis and tumor invasion (2). Structurally, MMP-14 consists of the following domains: a pro domain containing the furin cleavage site, a catalytic domain containing the zinc-binding site, a hinge region, four hemopexin-like domains, a transmembrane domain, and a cytoplasmic tail (3). Recombinant human (rh) MMP-14, consists of the pro domain, catalytic domain, hinge region and hemopexin-like domains. Because it lacks the transmembrane domain, rhMMP-14 is secreted and purified as a soluble protein. The pro domain was engineered to contain an Arg108Pro mutation, which prevents activation by furin cleavage. Activation of rhMMP-14 occurs by treatment with rhTrypsin 3 as described in the Activity Assay Protocol.
- Seiki, M. (2003) Cancer Lett. 194:1.
- Friedl, P. and K. Wolf (2008) Cancer Res. 68:7247.
Citation for Recombinant Human MMP-14/MT1-MMP (NS0-expressed) Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
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Extracellular Matrix Remodeling by Fibroblast-MMP14 Regulates Melanoma Growth
Authors: E Pach, M Kümper, JE Fromme, J Zamek, F Metzen, M Koch, C Mauch, P Zigrino
International Journal of Molecular Sciences, 2021-11-12;22(22):.
Species: Human
Sample Types: Recombinant Proteins
Applications: Bioassay
FAQs
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Can the enzyme be stored after activation, or do I need to use it immediately after activation?
We recommend only activating the amount of enzyme needed for your assay, and recommend activating the enzyme immediately prior to use. Any unactivated enzyme should be stored in aliquots at either the stock concentration at which the enzyme was supplied, or the reconstitution concentration, according to the product datasheet.
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Does this MMP enzyme need to be activated to work?
Yes, this enzyme requires activation prior to use.
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What is the activity of this enzyme in units/µg?
We supply this enzyme as a mass and calculate its activity relative to mass (pmol/min/µg). We have not calibrated this enzyme to an international standard unit, so we are unable to provide a conversion to units/µg.
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