Recombinant Cynomolgus Monkey MMP-9 Protein, CF Summary
Product Specifications
Ala20-Asp707
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.
10833-MP
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, CaCl2, NaCl and Brij-35. |
| Shipping | The product is shipped with dry ice or equivalent. 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% Brij-35 (w/v), pH 7.5 (TCNB)
- Recombinant Cynomolgus Monkey MMP-9 (rcynoMMP-9) (Catalog # 10833-MP)
- p-aminophenylmercuric acetate (APMA) (Sigma, Catalog # A9563), 100 mM stock in DMSO
- Substrate: Mca-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH2 (Catalog # ES001), 2 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rcynoMMP-9 to 100 µg/mL in Assay Buffer.
- Activate rcynoMMP-9 by adding APMA to a final concentration of 1 mM.
- Incubate at 37 °C for 24 hours.
- Dilute activated rcynoMMP-9 to 0.2 µg/mL in Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- Load 50 µL of 0.2 µg/mL rcynoMMP-9 into a plate and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 20 µM Substrate.
- 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)
- rcynoMMP-9: 0.01 µg
- Substrate: 10 µM
Scientific Data
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Recombinant Cynomologous Monkey MMP-9 Protein (Catalog # 10833-MP) is measured by its ability to cleave the fluorogenic peptide substrate, Mca-PLGL-Dpa-AR-NH2 (ES001).
View Larger
2 μg/lane of Recombinant Cynomolgus Monkey MMP-9 (Catalog # 10833-MP) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at ~90 kDa under reducing conditions.
Reconstitution Calculator
Background: MMP-9
Matrix metalloproteinase 9 (MMP-9), also known as gelatinase B, is a member of the MMP zinc-dependent family of endopeptidases. It cleaves and degrades a variety of targets including important extracellular matrix (ECM) proteins: gelatin, collagen, and elastin, as well as chemokines and extracellular domain plasma membrane proteins (1-3). MMP-9 is synthesized and secreted by several cells including neutrophils, macrophages, fibroblasts, and endothelial cells (4). The monomeric MMP-9 protein is composed of several distinct domains including a signal sequence, a pro-domain which is cleaved upon activation, and a catalytic domain at the n-terminus followed by a hinge region and the c-terminal hemopexin-like domains that contribute to substrate recognition and specificity (5,6). The catalytic domain contains fibronectin type II domains, an active site, and a zinc binding site. MMP-9 can exist as a monomer, disulfide-linked homodimer, or heterodimer in complex with lipocalin‑2 (7,8). MMP-9 activity is regulated at several levels via transcription, post-transcription, translation, secretion, activation, and inhibition. As MMP-9 is involved in ECM remodeling and membrane protein cleavage, it has been widely associated to play a role in several diseases including cancers (9), autoimmune, and cardiovascular diseases (9-11). MMP-9 is consequently an important target of interest for inhibition (11-13). Additionally, it has been found to be a potential biomarker for many types of cancer including pancreatic, osteosarcoma, lung, ovarian, and breast (9).
- Kridel, S.J. et al. (2001) J. Biol. Chem. 276:20572.
- Vaisar, T. et al. (2009) Mol. Cell. Proteom. MCP 8:1044.
- Dufour, A. and C.M. Overall. (2013) Trends Pharmacol. Sci. 34:233.
- Vandooren, J. et al. (2013) Crit. Rev. Biochem. Mol. Biol. 48:222.
- Roeb, E. et al. (2002) J. Biol. Chem. 277:50326.
- Rosenblum, G. et al. (2007) Structure 15:1227.
- Kjeldsen, L. et al. (1993) J. Biol. Chem. 268:10425.
- Olson, M.W. et al. (2000) J. Biol. Chem. 275:2661.
- Huang, H. (2018) Sensors 18:3249.
- Ram, M. et al. (2006) J. Clin. Immunol. 26:299.
- Hu, J. et al. (2007) Nat. Rev. Drug. Discov. 6:480.
- Fields, G.B. (2019) Cells. 8:984.
- Kumar, G.B. et al. (2019) Medchemcomm. 10:2024.
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