Recombinant Rat MMP-8 Protein, CF Summary
Product Specifications
Leu21-Pro466, with a C-terminal 6-His tag
Analysis
Product Datasheets
3245-MP
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.
3245-MP
| Formulation | Supplied as a 0.2 μm filtered solution in MES, NaCl and CaCl2. |
| 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
- Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (v/v) Brij-35, pH 7.5 (TCNB)
- Recombinant Rat MMP‑8 (rrMMP-8) (Catalog # 3245-MP)
- p-aminophenylmercuric acetate (APMA) (Sigma, Catalog # A-9563), 100 mM stock in DMSO
- ZnCl2, 1 M 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
- Dilute rrMMP-8 to 100 µg/mL in Assay Buffer.
- Activate rrMMP-8 by adding APMA and ZnCl2 to final concentrations of 1 mM and 25 µM respectively.
- Incubate at room temperature for 4 hours.
- Dilute activated rrMMP-8 to 2 ng/μL in Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- Load 50 µL of the 2 ng/µL rrMMP-8 into in 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.
- 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).
- rrMMP-8: 0.10 µg
- Substrate: 10 µM
Background: MMP-8
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. MMP-8 (neutrophil collagenase, collagenase 2) is expressed in neutrophils, where it is stored in specific granules. MMP-8 release from the neutrophils is stimulated by various factors such as interleukins 1 and 8, TNF-alpha and GM-CSF. MMP-8 is capable of cleaving types I, II and III triple-helical collagen, gelatin peptides, fibronectin, proteoglycans, aggrecan, serpins, beta -casein and peptides such as angiotensin and substance P. In addition to its function in phagocytosis, MMP-8 has a high capacity for infiltrating connective tissue, and is implicated in the breakdown of the extracellular matrix in diseases such as rheumatoid arthritis. Structurally, MMP-8 consists of several domains: a pro-domain that is cleaved upon activation, a catalytic domain containing the zinc-binding site, a short hinge region and a hemopexin-like domain (1).
- H. Tschesche et al. (2004) Handbook of Proteolytic Enzymes (eds. A.J. Barrett et al.) p.480, Academic Press, San Diego.
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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