Recombinant Human MMP-16/MT3-MMP Protein, CF Summary
Ala32-Gly291 (Ile152Asn), with a C-terminal 10-His tag
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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.
|Formulation||Supplied as a 0.2 μm filtered solution in Tris, NaCl, CaCl2, ZnCl2 and 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.
- Activation Buffer: 50 mM Tris, 1 mM CaCl2, 0.5% (v/v) Brij-35, pH 9.0
- Assay Buffer: 50 mM Tris, 3 mM CaCl2, 1 µM ZnCl2, pH 8.5
- Recombinant Human MMP‑16/MT3‑MMP (rhMMP-16) (Catalog # 1785-MP)
- Recombinant Human Furin (rhFurin) (Catalog # 1503-SE)
- Substrate: MCA-Lys-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH2 (Catalog # ES010)
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhMMP-16 to 80 µg/mL in Activation Buffer.
- Dilute rhFurin to 1.76 µg/mL in Activation Buffer.
- Mix equal volumes of 80 µg/mL of rhMMP-16 and 1.76 µg/mL of rhFurin together.
- Incubate at 37 °C for 1 hour to activate rhMMP-16.
- After incubation, dilute activated rhMMP-16 to 2 ng/µL in Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- In plate, load 50 µL of 2 ng/µL rhMMP-16, and start the reaction by adding 50 µL of 20 µM Substrate to wells. As a Substrate Blank, load 50 µL of Assay Buffer and 50 µL of 20 µM Substrate.
- Read at excitation and emission wavelengths of 320 nm and 405 nm, 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-16: 0.1 µg
- Substrate: 10 µM
Matrix metalloproteinases (MMPs) are a family of zinc and calcium dependent endopeptidases with the combined ability to degrade all the components of the extracellular matrix (ECM). MMP‑16 (MT3-MMP) is found in brain, lung, placenta, smooth muscle cells, and malignant tumor tissues including oral melanoma and renal carcinoma (1). MMP‑16 has been shown to activate proMMP-2 and degrade various ECM components including native collagens (2, 3). MMP‑16 has been proposed to possess the potential to directly enhance the growth and invasiveness of cells in vivo, two critical processes for development and carcinogenesis (4). Structurally, MMP‑16 consists of the following domains: a pro domain containing the furin cleavage site, a catalytic domain containing the zinc-binding site, a hinge region, a hemopexin-like domain, a transmembrane domain, and a cytoplamasic tail (1). The structure of the catalytic domain in complex with a hydroxamate inhibitor has been solved (5). The recombinant human MMP‑16PC consists of the pro and catalytic domains, which can be activated by treatment with furin.
- Takino, T. et al. (1995) J. Biol. Chem. 270:23013.
- Shofuda, K. et al. (1997) J. Biol. Chem. 272:9749.
- Shimada, T. et al. (1999) Eur. J. Biochem. 262:907.
- Kang, T. et al. (2000) FASEB J. 14:2559.
- Lang, R. et al. (2004) J. Mol. Biol. 336:213.
Citations for Recombinant Human MMP-16/MT3-MMP 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.
Citations: Showing 1 - 3
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Kallikrein-Related Peptidase 14 Activates Zymogens of Membrane Type Matrix Metalloproteinases (MT-MMPs)-A CleavEx Based Analysis
Authors: K Falkowski, E Bielecka, IB Thøgersen, O Boche?ska, K P?aza, M Kali?ska, L S?siadek, M Magoch, A P?cak, M Wi?niewska, N Gruba, M Wysocka, A Wojtysiak, M Brzezi?ska, K Sychowska, A Pejkovska, M Rehders, G Butler, CM Overall, K Brix, G Dubin, A Lesner, A Kozik, JJ Enghild, J Potempa, T Kantyka
Int J Mol Sci, 2020;21(12):.
Sample Types: Recombinant Protein
Discovery of potent and specific inhibitors targeting the active site of MMP-9 from the engineered SPINK2 library
Authors: H Yano, D Nishimiya, Y Kawaguchi, M Tamura, R Hashimoto
PLoS ONE, 2020;15(12):e0244656.
Sample Types: Whole Cells
Astacin proteases cleave dentin sialophosphoprotein (Dspp) to generate dentin phosphoprotein (Dpp).
Authors: Tsuchiya S, Simmer JP, Hu JC, Richardson AS, Yamakoshi F, Yamakoshi Y
J. Bone Miner. Res., 2010;26(0):220.
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.
Does this MMP enzyme need to be activated to work?
Yes, this enzyme requires activation prior to use.
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.
Fluorogenic Peptide Substrates
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