Recombinant Human MMP-15/MT2-MMP Protein, CF Summary
Product Specifications
Glu47-Pro565 (Arg128Pro) (Arg129Gly), with a C-terminal 5-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.
916-MP
| Formulation | Supplied as a 0.2 μm filtered solution in MES, NaCl 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.
|
Assay Procedure
- Activation Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 5 µM ZnCl2, 0.05% (w/v) Brij-35, pH 7.5
- Assay Buffer: 50 mM Tris, 500 mM NaCl, 5 mM CaCl2, 1 µM ZnCl2, 0.02% (w/v) Brij-35, pH 8.0
- Recombinant Human MMP‑15/MT2‑MMP (rhMMP‑15) (Catalog # 916-MP)
- Recombinant Human Active Trypsin 3/PRSS3 (rhTrypsin 3) (Catalog # 3714-SE) )
- 4-(2-Aminoethyl-benzensulfonyl fluoride hydrochloride) (AEBSF) (Tocris, Catalog # 5175), 100 mM stock in deionized water
- 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
- Activate rhMMP-15 at 100 µg/mL with 0.1 µg/mL rhTrypsin 3 in Activation Buffer.
- Incubate at 37 °C for 2 hours.
- Add AEBSF for a final concentration of 1 mM and incubate at room temperature for 15 minutes to stop the reaction.
- Dilute activated rhMMP-15 to 1 ng/µL in Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- Load 50 µL of the 1 ng/µL rhMMP-15 in a black well plate, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 20 µM Substrate without any rhMMP-15.
- 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-15: 0.05 µg
- Substrate: 10 µM
Reconstitution Calculator
Background: MMP-15/MT2-MMP
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. They play critical roles in tissue remodeling, angiogenesis, tumor invasion, and rheumatoid arthritis (1). MMP-15, also known as MT2-MMP, is a membrane-type MMP that is expressed in many tumor tissues including urothelial carcinoma, oral cancer, ovarian carcinoma, melanoma, and astrocytoma (2). Structurally, MMP-15 consists of the following domains:a pro domain containing a furin cleavage site, a catalytic domain containing the zinc-binding site, a hinge region, a hemopexin-like domain, a transmembrane domain, and a cytoplasmic tail (1). Recombinant Human (rh) MMP-15, consists of the pro domain, catalytic domain, hinge region and hemopexin-like domain. The pro domain contains the mutations R128P and R129G, which prevent activation by furin cleavage. Activation of rhMMP-15 is possible by treatment with rhTrypsin 3 as described in the Activity Assay Protocol.
- Takino, T. et al. (1995) J. Biol. Chem. 270:23013.
- Yana I. and M. Seiki (2004) Handbook of Proteolytic Enzymes (ed. Barrett, et al.) p. 549-551, Academic Press, San Diego.
Product Specific Notices
Coomassie is a registered trademark of Imperial Chemical Industries Ltd.Citation for Recombinant Human MMP-15/MT2-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.
1 Citation: Showing 1 - 1
-
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, 2020-06-19;21(12):.
Species: Human
Sample Types: Recombinant Protein
Applications: Bioassay
FAQs
-
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.
Reviews for Recombinant Human MMP-15/MT2-MMP Protein, CF
There are currently no reviews for this product. Be the first to review Recombinant Human MMP-15/MT2-MMP Protein, CF and earn rewards!
Have you used Recombinant Human MMP-15/MT2-MMP Protein, CF?
Submit a review and receive an Amazon gift card.
$25/€18/£15/$25CAN/¥75 Yuan/¥2500 Yen for a review with an image
$10/€7/£6/$10 CAD/¥70 Yuan/¥1110 Yen for a review without an image
