Tissue inhibitors of metalloproteinases or TIMPs are a family of homologous proteins that regulate the activity of matrix metalloproteinases (MMPs) (1, 2). There are four known members of the family, TIMP‑1, TIMP-2, TIMP-3 and TIMP-4 that have been found to exhibit multiple functions, including inhibition of active MMPs, proMMP activation, cell growth promotion, matrix binding, inhibition of angiogenesis and the induction of apoptosis. Structurally, TIMPs have two domains, an N‑terminal domain and a C‑terminal domain. Each domain consists of three disulfide-bonded loops. TIMP-1 is a glycoprotein produced by a wide range of cell types. Through its N‑terminal domain, TIMP-1 inhibits active MMPs by forming a non-covalent binary complex with the MMP active site. The C-terminal domain of TIMP-1 interacts with the C-terminal domain of proMMP-9, which may play a role in regulating proMMP-9 activation.
Recombinant Mouse TIMP-1 Protein, CF
R&D Systems | Catalog # 980-MT
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Key Product Details
- R&D Systems NS0-derived Recombinant Mouse TIMP-1 Protein (980-MT)
- Quality control testing to verify active proteins with lot specific assays by in-house scientists
- All R&D Systems proteins are covered with a 100% guarantee
Source
NS0
Accession Number
Applications
Inhibition Activity
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Product Specifications
Source
Mouse myeloma cell line, NS0-derived mouse TIMP-1 protein
Cys25-Arg205
Cys25-Arg205
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
N-terminal Sequence Analysis
Cys25
Predicted Molecular Mass
20 kDa
SDS-PAGE
32 kDa, reducing conditions
Activity
Measured by its ability to inhibit human MMP-2 cleavage of a fluorogenic peptide substrate Mca-PLGL-Dpa-AR-NH2 (Catalog # ES001).
The IC50 value is <3.5 nM, as measured under the described conditions.
Reviewed Applications
Read 2 reviews rated 4 using 980-MT in the following applications:
Formulation, Preparation, and Storage
980-MT
| Formulation | Lyophilized from a 0.2 μm filtered solution in Tris and NaCl. |
| Reconstitution | Reconstitute at 100 μg/mL in sterile 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, and 0.05% Brij-35, pH 7.5. |
| 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.
|
Calculators
Background: TIMP-1
References
- Murphy, G. and F. Willenbrock (1995) Methods Enzymol. 248:496.
- Brew, K. et al. (2000) Biochim. Biophys. Acta 1477:267.
Long Name
Tissue Inhibitors of Metalloproteinases 1
Alternate Names
TIMP1
Gene Symbol
TIMP1
UniProt
Additional TIMP-1 Products
Product Documents for Recombinant Mouse TIMP-1 Protein, CF
Certificate of Analysis
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Note: Certificate of Analysis not available for kit components.
Product Specific Notices for Recombinant Mouse TIMP-1 Protein, CF
For research use only
Related Research Areas
Citations for Recombinant Mouse TIMP-1 Protein, CF
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Application: MTT assayVerified Customer | Posted 09/20/2018
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Protocols
View specific protocols for Recombinant Mouse TIMP-1 Protein, CF (980-MT):
Materials
- Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35 (w/v), pH 7.5 (TCNB)
- Recombinant Mouse TIMP-1 (rmTIMP-1) (Catalog # 980-MT)
- Recombinant Human MMP‑2 (rhMMP‑2) (Catalog # 902-MP)
- p-aminophenylmercuric acetate (APMA), (Sigma, Catalog # A-9563), 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)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhMMP-2 to 100 µg/mL in Assay Buffer containing 1 mM APMA.
- Incubate at 37 °C for 1 hour (activation).
- Prepare a curve of rmTIMP-1 (MW: 20,200 Da). Make the following serial dilutions in Assay Buffer: 5000, 2000, 1000, 500, 300, 200, 150, 100, 20, and 2 nM.
- Dilute activated rhMMP-2 to 12.5 µg/mL in Assay Buffer.
- Mix 25.6 µL of diluted rhMMP-2, 16 µL of rmTIMP-1 serial curve dilutions, and 118.4 µL of Assay Buffer.
- Include two enzyme controls of 25.6 µL of diluted rhMMP-2 and 134.4 μL Assay Buffer.
- Incubate reaction mixtures at 37 °C for 2 hours.
- Dilute incubated reaction mixtures by a 5-fold dilution in Assay Buffer.
- Dilute Substrate to 10 µM in Assay Buffer.
- In a plate load 50 µL of the diluted incubated reaction mixtures to wells.
- Start the reaction by adding 50 µL of 10 µM Substrate to wells.
- 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-1 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-Pro-Leu-OH (Bachem, Catalog # M-1975).
Per Well:
- rhMMP-2: 0.02 µg
- rmTIMP-1: 50, 20, 10, 5, 3, 2, 1.5, 1, 0.2, and 0.02 nM
- Substrate: 5 µM