详细说明
- Purity>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
- Endotoxin Level<0.10 EU per 1 μg of the protein by the LAL method.
- ActivityMeasured by its ability to cleave the fluorogenic peptide substrate, Mca-PLGL-Dpa-AR-NH 2 (Catalog # ). The specific activity is >500 pmol/min/μg, as measured under the described conditions.
- SourceMouse myeloma cell line, NS0-derived Leu17-Cys470, with Val97Leu substitution
- Accession #
- N-terminal Sequence
AnalysisLeu17 - Structure / FormPro form
- Predicted Molecular Mass52 kDa
- SDS-PAGE53-60 kDa, reducing conditions
| 917-MPB | | |
| Formulation Lyophilized from a 0.2 μm filtered solution in MES, NaCl, CaCl 2, CHAPS, ZnSO 4 and PEG. | ||
| Reconstitution Reconstitute at 0.25 mg/mL in 10 mM MES, 0.1 M NaCl, 100 μM CaCl 2, 0.1% CHAPS, 1 μM ZnSO 4 and 0.1% PEG., pH 6.0. | ||
| 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
Materials
- Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
- Recombinant Human MMP-12 (rhMMP-12) (Catalog # 917-MPB)
- p-aminophenylmercuric acetate (APMA) (Sigma, Catalog # A9563), 100 mM stock in DMSO
- Substrate MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH2 (Catalog # ), 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 rhMMP-12 to 50 µg/mL in Assay Buffer.
- Activate rhMMP-12 by adding APMA to a final concentration of 1 mM and incubating at 37 °C for 24 hours.
- Dilute activated rhMMP-12 to 0.4 ng/µL in Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- Load into plate 50 µL of 0.4 ng/µL rhMMP-12, 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).
Per Well:
- rhMMP-12: 0.020 µg
- Substrate: 10 µM
Background: MMP-12
Matrix metalloproteinases (MMPs) are a family of zinc and calcium dependent endopeptidases that collectively degrade the components of the extracellular matrix. MMP-12 (macrophage elastase) consists of the following domains: a pro domain, a catalytic domain containing the zinc-binding site, and a C-terminal hemopexin-like domain. The 54 kDa rhMMP-12 pro form is activated via processing into 45 kDa and 22 kDa active forms (1). MMP-12 is capable of cleaving several substrates in addition to elastin. Macrophage secretion of MMP-12 at sites of inflammation can be induced by cytokines. Given the secretion of MMP12 during an inflammatory response, MMP-12 is involved in many pathologies including vascular disease (2, 3) and cancer (4-6). In particular, MMP12-mediated pathological degradation of the extracellular matrix is a well-established key event in inflammatory-related pulmonary disease (7). For example, overexpression of MMP12 in alveolar macrophages is associated with smoking and emphysema (8) while different MMP-12 variants result in protection against chronic obstructive pulmonary disease (COPD) development (9) or cause increased risk and disease severity (10). MMP-12 has also been shown to translocate into the nucleus of viral-infected cells to directly regulate transcription during an immune response (11).
- References:
- Shapiro, S.D. et al. (1993) J. Biol. Chem. 268:23824.
- Liu, S.L. et al. (2015) Sci. Rep. 5:17189.
- Iyer, R.P. et al. (2015) Int. J. Cardiol. 185:198.
- Cao, W. et al. (2017) Oncol. Rep. 3:1401.
- Klupp, F. et al. (2016) BMC Cancer 16:494.
- Chung, I.C. et al. (2014) BMC Cancer 14:348.
- Vanderbroucke, R.E. et al. (2011) Eur. Respir. J. 387:1200.
- Babusyte, A. et al. (2007) Respir. Res. 8:81.
- Hunninghake, G.M. et al. (2009) N. Engl. J. Med. 361:2599.
- Mukhopadhyay, S. et al. (2010) J. Allergy Clin. Immunol. 126:70.
- Marchant, D.J. et al. (2013) Nature Medicine 20:493.
- Long Name:Matrix Metalloproteinase 12
- Entrez Gene IDs:4321 (Human); 17381 (Mouse)
- Alternate Names:EC 3.4.24; hME; HMEEC 3.4.24.65; Macrophage elastase; macrophage metalloelastase; matrix metallopeptidase 12 (macrophage elastase); matrix metalloproteinase 12 (macrophage elastase); Matrix metalloproteinase-12; ME; MGC138506; MME; MMP12; MMP-12
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