Recombinant Mouse MMP-12 Protein, CF 20 UG_行业前沿_产品

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.
Activity
Measured 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 .
Source
Mouse myeloma cell line, NS0-derived Ala18-Cys462
Accession #
N-terminal Sequence
Analysis
Ala18
Structure / Form
Pro form
Predicted Molecular Mass
52 kDa
SDS-PAGE
50-62 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. 6 months from date of receipt, -20 to -70 °C as supplied. 3 months, -20 to -70 °C under sterile conditions after reconstitution.

Assay Procedure

Materials

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 V_max* (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

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 52 kDa rmMMP-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). Recently, MMP-12 has 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