Recombinant Human MMP-2 (NS0-expressed) Protein, CF Summary
Product Specifications
Ala30-Cys660
Analysis
Product Datasheets
902-MPN
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.
902-MPN
| Formulation | Lyophilized from a 0.2 μm filtered solution in Tris, CaCl2, NaCl and ZnCl2. |
| Reconstitution | Reconstitute at 100 μg/mL in sterile 100 mM Tris, 10 mM CaCl2, 150 mM NaCl, and 0.05% Brij-35, pH 8.0. |
| Shipping | The product is shipped with polar packs. 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: 100 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 8.0
- Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij 35, pH 7.5 (TCNB)
- Recombinant Human MMP-2 (rhMMP-2) (Catalog # 902-MPN)
- p-aminophenylmercuric acetate (APMA), (Sigma, Catalog # A-9563), prepare a 100 mM stock in DMSO
- Fluorogenic Peptide Substrate I: MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH2 (Catalog # ES001), 1 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhMMP-2 to 100 µg/mL in Activation Buffer.
- Activate rhMMP-2 by adding APMA to a final concentration of 1 mM.
- Incubate at 37 °C for 1 hour.
- Dilute activated rhMMP-2 to 0.2 ng/µL in assay buffer.
- Dilute substrate to 20 µM in assay buffer.
- Load in plate 50 µL of the 0.2 ng/µL rhMMP-2 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.
- 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-2: 0.010 µg
- Substrate: 10 µM
Background: MMP-2
Matrix metalloproteinases are a family of zinc and calcium dependent endopeptidases with the combined ability to degrade all the components of the extracellular matrix. MMP‑2 (gelatinase A), a type IV collagenase, can degrade a broad range of substrates including type IV, V, VII and X collagens as well as elastin and fibronectin. It is believed to act synergistically with interstitial collagenase (MMP‑1) in the degradation of fibrillar collagens as it degrades their denatured gelatin forms. MMP‑2 has been shown to be associated with many connective tissue cells as well as neutrophils, macrophages and monocytes. Structurally, MMP‑2 may be divided into several distinct domains: a pro-domain which is cleaved upon activation; a catalytic domain containing the zinc binding site; a fibronectin-like domain thought to play a role in substrate targeting; and a carboxyl terminal (hemopexin-like) domain containing 2 N-linked glycosylation sites.
Citation for Recombinant Human MMP-2 (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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Angiotensin II-accelerated atherosclerosis and aneurysm formation is attenuated in osteopontin-deficient mice.
Authors: Bruemmer D, Collins AR, Noh G, Wang W, Territo M, Arias-Magallona S, Fishbein MC, Blaschke F, Kintscher U, Graf K, Law RE, Hsueh WA
J. Clin. Invest., 2003-11-01;112(9):1318-31.
Applications: Zymography
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 enzyme have a tag?
No, this enzyme does not have a tag. Please refer to the Source section on the product-specific page or product datasheet for sequence information.
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If I use this enzyme at a higher concentration, do I need to change the concentration of APMA to activate it?
We have only optimized activation conditions for one particular concentration of this MMP enzyme as part of our regular QC testing for enzymatic activity. Activating the enzyme at any different concentration would have to be optimized by the end user.
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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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