Recombinant Rat MMP-9 Protein, CF Summary
Product Specifications
Leu35-Pro708, with a C-terminal 6-His tag
Analysis
Product Datasheets
5427-MM
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.
5427-MM
| Formulation | Supplied as a 0.2 μm filtered solution in MES, NaCl, CaCl2 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
- Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
- Recombinant Rat MMP-9 (rrMMP-9) (Catalog # 5427-MM)
- p-aminophenylmercuric acetate (APMA) (Sigma, Catalog # A-9563), 100 mM stock in DMSO
- Substrate: MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH2 (Catalog # ES001), 2 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rrMMP-9 to 100 µg/mL in Assay Buffer.
- Activate rrMMP-9 by adding APMA to a final concentration of 1 mM.
- Incubate activation reaction at 37 °C for 8 hours.
- Dilute activated rrMMP-9 to 0.2 ng/µL in Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- In a plate load 50 µL of 0.2 ng/µL rrMMP-9, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank of 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).
Background: MMP-9
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-9 (gelatinase B) can degrade a broad range of substrates including gelatin, collagen types IV and V, elastin and proteoglycan core protein. 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-9 is produced by keratinocytes, monocytes, macrophages and PMN leukocytes. MMP-9 is present in most cases of inflammatory responses. Structurally, MMP-9 may be divided into five distinct domains: a pro-domain which is cleaved upon activation, a catalytic domain containing the zinc binding site, a gelatin-binding domain consisting of three contiguous fibronectin type II units, a proline-rich linker region, and four carboxyl terminal hemopexin-like domains.
Citations for Recombinant Rat MMP-9 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.
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Citations: Showing 1 - 4
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Homeostatic regulation of perisynaptic MMP9 activity in the amblyopic visual cortex
Authors: S Murase, D Winkowski, J Liu, PO Kanold, EM Quinlan
Elife, 2019-12-23;8(0):.
Species: Mouse
Sample Types: Protein
Applications: Bioassay -
MMP Inhibition Preserves Integrin Ligation and FAK Activation to Induce Survival and Regeneration in RGCs Following Optic Nerve Damage
Authors: PM D'Onofrio, AP Shabanzade, BK Choi, M Bähr, PD Koeberle
Invest. Ophthalmol. Vis. Sci., 2019-02-01;60(2):634-649.
Species: Rat
Sample Types: Recombinant Protein
Applications: Western Blot -
Matrix Metalloproteinase-Mediated Blood-Brain Barrier Dysfunction in Epilepsy
Authors: RG Rempe, AMS Hartz, ELB Soldner, BS Sokola, SR Alluri, EL Abner, RJ Kryscio, A Pekcec, J Schlichtig, B Bauer
J. Neurosci., 2018-04-09;0(0):.
Species: Rat
Sample Types:
Applications: Zymography -
Cannabinoid CB2 receptors regulate central sensitization and pain responses associated with osteoarthritis of the knee joint.
Authors: Burston, James J, Sagar, Devi Ran, Shao, Pin, Bai, Mingfeng, King, Emma, Brailsford, Louis, Turner, Jenna M, Hathway, Gareth J, Bennett, Andrew J, Walsh, David A, Kendall, David A, Lichtman, Aron, Chapman, Victoria
PLoS ONE, 2013-11-25;8(11):e80440.
Species: Rat
Sample Types: Tissue Homogenates
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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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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