Recombinant Mouse TIMP-4 Protein, CF
Recombinant Mouse TIMP-4 Protein, CF Summary
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.
|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 Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35, pH 7.5 (TCNB)
- Recombinant Mouse TIMP-4 (rmTIMP-4) (Catalog # 7667-TM)
- Recombinant Human MMP‑2 (rhMMP-2) (Catalog # 902-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)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhMMP-2 to 100 µg/mL in Assay Buffer with 1 mM APMA.
- Incubate at 37 °C for 1 hour.
- Prepare a curve of rmTIMP-4 (MW: 22,600 Da). Make the following serial dilutions in Assay Buffer: 4000, 2000, 1000, 500, 250, 125, 62.5, 31.3, 15.6, and 7.8 nM.
- Dilute activated rhMMP-2 to 12.5 µg/mL in Assay Buffer.
- For each inhibition point, mix 25.6 µL of 12.5 µg/mL rhMMP-2, 16 µL of the rmTIMP-4 curve dilution, and 118.4 µL of Assay Buffer. Include two enzyme controls of 25.6 µL of 12.5 µg/mL rhMMP-2 and 134.4 µL Assay Buffer.
- Incubate reaction mixtures at 37 °C for 2 hours.
- Dilute incubated reaction mixtures 5-fold in Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- Load 50 µL of the incubated reactions into the wells of a black well plate. Start the reaction by adding 50 µL of 20 µM Substrate.
- Read at excitation and emission wavelengths of 320 nm and 405 nm (top read) in kinetic mode for 5 minutes.
- Derive the 50% inhibiting concentration of rmTIMP-4 (IC50) 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).
- rhMMP-2: 0.02 µg (2.82 nM)
- rmTIMP-4: 40, 20, 10, 5, 2.5, 1.25, 0.63, 0.31, 0.16, and 0.078 nM
- Substrate: 10 µM
Tissue inhibitors of metalloproteinases (TIMPs) are a family of secreted proteins that regulate the activation and proteolytic activity of the zinc enzymes known as matrix metalloproteinases (MMPs). There are four known members of the family, TIMP-1, -2, -3, and -4. TIMP-4 is produced by a wide range of tissues, particularly brain, heart, ovary and skeletal muscle (1, 2). Limited studies have shown that TIMP-4 has a tumor suppressive effect against Wilm’s tumor, exhibits negative correlation with glioma maligancy and is found in breast carcinoma cells (3, 5). TIMP-4 inhibits MMP-mediated proteolysis by forming a noncovalent binary complex with the MMP active site through its N-terminal domain. In addition, it binds to the hemopexin-like domain of proMMP-2 through its C-terminal domain in a manner similar to TIMP-2 (6). However, unlike TIMP-2, TIMP-4 does not promote proMMP-2 activation by MT1-MMP (MMP-14) (7). Although TIMP-4 is a potent inhibitor of most MMPs, it is not an effective inhibitor of ADAMs, such as TACE (8, 9).
- Greene et al. (1996) J. Biol. Chem. 271:30375.
- Leco et al. (1997) FEBS Lett. 401:213.
- Geliker et al. (2001) Oncogene 20:4337.
- Groft et al. (2001) Br. J. Cancer 85:55.
- Hurst et al. (2001) Biochem. Biophys. Res. Comm. 281:166.
- Bigg et al. (1997) J. Biol. Chem. 272:15496.
- Hernandez-Barrantes et al. (2001) Biochem. Biophys. Res. Comm. 281:126.
- Amour et al. (1998) FEBS Lett. 435:39.
- Liu et al. (1997) J. Biol. Chem. 272:20479.
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Fluorogenic Peptide Substrates
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