Recombinant Mouse TIMP-2 Protein, CF Summary
Product Specifications
Met1-Pro220
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.
6304-TM
| Formulation | Supplied as a 0.2 μm filtered solution in Tris and NaCl. |
| 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
- Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
- Recombinant Mouse TIMP-2 (rmTIMP-2) (Catalog # 6304-TM)
- Recombinant Human MMP‑2 (rhMMP-2) (Catalog # 902-MP)
- 4-Aminophenylmercuric acetate (APMA) (Sigma, Catalog # A9563), 40 mM stock in DMSO
- Substrate: MCA-PLGL-DPA-AR-NH2 (Catalog # ES001), 2 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: Gemini EM by Molecular Devices) or equivalent
- Dilute rhMMP-2 to 100 µg/mL in Assay Buffer.
- Add APMA to the rhMMP-2 to a final concentration of 1 mM.
- Incubate the 100 µg/mL rhMMP-2 at 37 °C for 1 hour to activate.
- Prepare a curve of rmTIMP-2 (MW = 21,700 kDa) in Assay Buffer. Make the following serial dilutions: 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, and 1.56 nM.
- Dilute the activated rhMMP-2 to 2 µg/mL in Assay Buffer.
- Combine 20 µL of each dilution with 20 µL of the 2 µg/mL rhMMP-2. Include an enzyme control containing Assay Buffer in place of rmTIMP-2.
- Incubate reaction mixtures at 37 °C for two hours.
- Dilute reactions by adding 160 µL Assay Buffer to each.
- Dilute Substrate to 20 µM in Assay Buffer.
- Load into a black well plate 50 µL of the diluted incubated mixtures, and start the reaction by adding 50 µL substrate.
- Read at excitation and emission wavelengths of 320 nm and 405 nM (top read), respectively, in kinetic mode for 5 minutes.
- Derive the 50% inhibiting concentration (IC50) for rmTIMP-2 by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
- The specific activity for rhMMP-2 at each point may be determined using the following formula (if needed):
|
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-P-L-OH (Bachem, Catalog # M-1975).
- rhMMP-2: 0.01 µg
- rmTIMP-2: 20 nM, 10 nM, 5 nM, 2.5 nM, 1.25 nM, 0.625 nM, 0.313 nM, 0.156 nM, 0.078 nM
- Substrate: 10 µM
Reconstitution Calculator
Background: TIMP-2
Tissue inhibitors of metalloproteinases (TIMPs) are a family of proteins that regulate the activation and proteolytic activity of matrix metalloproteinases (MMPs). There are four mammalian members of the family; TIMP‑1, TIMP‑2, TIMP‑3, and TIMP‑4. TIMP‑2 is a non‑glycosylated protein of molecular mass 22 kDa that is secreted by a wide range of cell types that inhibits MMPs non‑covalently by the formation of binary complexes. TIMP‑2 interacts with MMP‑14 to facilitate the cell‑surface activation of pro‑MMP‑2 (1). TIMP‑2 has other functions independent of the inhibition of MMPs. The binding of TIMP‑2 to a3b1 integrin at the surface of endothelial cells results in the inhibition of endothelial cell proliferation and angiogenesis (2).
Product Specific Notices
Coomassie is a registered trademark of Imperial Chemical Industries Ltd.Citations for Recombinant Mouse TIMP-2 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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Tissue Inhibitor of Metalloproteinase-1 Promotes Polymorphonuclear Neutrophil (PMN) Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces
Authors: X Wang, J Rojas-Quin, J Wilder, Y Tesfaigzi, D Zhang, CA Owen
J. Immunol., 2019-04-24;0(0):.
Species: Transgenic Mouse
Sample Types: Whole Cells
Applications: Bioassay -
Human umbilical cord plasma proteins revitalize hippocampal function in aged mice
Authors: JM Castellano, KI Mosher, RJ Abbey, AA McBride, ML James, D Berdnik, JC Shen, B Zou, XS Xie, M Tingle, IV Hinkson, MS Angst, T Wyss-Coray
Nature, 2017-04-19;544(7651):488-492.
Species: Mouse
Sample Types: In Vivo
Applications: In Vivo -
Cathepsin Protease Controls Copper and Cisplatin Accumulation via Cleavage of the Ctr1 Metal-binding Ecto-domain
J Biol Chem, 2016-05-03;0(0):.
Species: Mouse
Sample Types: Cell Lysates
Applications: Bioassay -
IgLON cell adhesion molecules are shed from the cell surface of cortical neurons to promote neuronal growth.
Authors: Sanz R, Ferraro G, Fournier A
J Biol Chem, 2014-12-23;290(7):4330-42.
Species: Rat
Sample Types: Whole Cells
Applications: Bioassay -
Activated hepatic stellate cells are dependent on self-collagen, cleaved by membrane type 1 matrix metalloproteinase for their growth.
Authors: Birukawa N, Murase K, Sato Y, Kosaka A, Yoneda A, Nishita H, Fujita R, Nishimura M, Ninomiya T, Kajiwara K, Miyazaki M, Nakashima Y, Ota S, Murakami Y, Tanaka Y, Minomi K, Tamura Y, Niitsu Y
J Biol Chem, 2014-05-27;289(29):20209-21.
Species: Rat
Sample Types: Whole Cells
Applications: Bioassay
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