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 a fluorogenic peptide substrate Mca-KPLGL-Dpa-AR-NH 2 (Catalog # ). The specific activity is >100 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .
- SourceMouse myeloma cell line, NS0-derived Ala18-Cys462
- Accession #
- N-terminal Sequence
AnalysisAla18 - Structure / FormPro form
- Predicted Molecular Mass52 kDa
- SDS-PAGE50-62 kDa, reducing conditions
3467-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 CaCl2, 0.1% CHAPS, 1 µM ZnSO4 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.
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- Activation Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5
(TCNB) + 5 µM ZnCl2 - Assay Buffer: 50 mM Tris, 10 mM CaCl2,150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
- Recombinant Mouse MMP-12 (rmMMP-12) (Catalog # 3467-MPB)
- p-aminophenylmercuric acetate (APMA) (Sigma, Catalog # A9563), 100 mM stock in DMSO
- Substrate: MCA-Lys-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: Gemini EM by Molecular Devices) or equivalent
- Dilute rmMMP-12 to 100 µg/mL with Activation Buffer containing 1 mM APMA.
- To activate, incubate rmMMP-12 at 37 °C for 24 hours.
- Dilute activated rmMMP-12 to 2 µg/mL in Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- Load 50 µL of 2 µg/mL rmMMP-12 to wells, 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.
- 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).
Per Well:- rmMMP-12: 0.1 µg
- Substrate: 10 µM
- 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