Recombinant Mouse MMP-12 Protein, CF Summary
Product Specifications
Ala18-Cys462
Analysis
Product Datasheets
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.
3467-MPB
| Formulation | Lyophilized from a 0.2 μm filtered solution in MES, NaCl, CaCl2, CHAPS, ZnSO4 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.
|
Assay Procedure
- 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 # ES010), 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
Reconstitution Calculator
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).
- 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.
Citations for Recombinant Mouse MMP-12 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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Preclinical development of SGN-CD47M: Protease-activated antibody technology enables selective tumor targeting of the innate immune checkpoint receptor CD47
Authors: Levengood, MR;Carosino, CM;Zhang, X;Lucas, S;Ortiz, DJ;Westendorf, L;Chin, AP;Martin, AD;Wong, A;Hengel, SM;Sun, H;Zeng, W;Yumul, R;Dominguez, MM;Chen, Y;Zheng, JH;Karlsson, CAB;Trang, VH;Senter, PD;Gardai, SJ;
Molecular cancer therapeutics
Species: N/A
Sample Types: Antibody
Applications: Bioassay -
MMP12 knockout prevents weight and muscle loss in tumor-bearing mice
Authors: L Jiang, M Yang, S He, Z Li, H Li, T Niu, D Xie, Y Mei, X He, L Wei, P Huang, M Huang, R Zhang, L Wang, J Li
BMC Cancer, 2021-12-04;21(1):1297.
Species: N/A
Sample Types: Peptide
Applications: Bioassay, Mass Spectrometry -
Elastolytic activity of cysteine cathepsins K, S, and V promotes vascular calcification
Authors: PM Andrault, P Panwar, NCW Mackenzie, D Brömme
Sci Rep, 2019-07-04;9(1):9682.
Species: Mouse
Sample Types: Whole Cells
Applications: Cell Culture -
Fibri(nogen) drives repair after acetaminophen-induced liver injury via leukocyte ?M?2 integrin-dependent upregulation of MMP12
Authors: Anna K Kopec
J. Hepatol, 2016-12-10;0(0):.
Species: Mouse
Sample Types: In Vivo
Applications: In Vivo
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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