Recombinant Human TIMP-4 (Sf 21-expressed) Protein, CF

R&D Systems | Catalog # 974-TSF

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Key Product Details

  • R&D Systems Sf 21 (baculovirus)-derived Recombinant Human TIMP-4 (Sf 21-expressed) Protein (974-TSF)
  • 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

Sf 21 (baculovirus)

Accession Number

Q99727

Applications

Inhibition Activity
View all TIMP-4 Proteins and Enzymes »

Product Specifications

Source

Spodoptera frugiperda, Sf 21 (baculovirus)-derived human TIMP-4 protein
Cys30-Pro224

Purity

>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Cys30

Predicted Molecular Mass

22 kDa

SDS-PAGE

23 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 approximately 2.5 nM, as measured under the described conditions.

Formulation, Preparation, and Storage

974-TSF
Formulation Lyophilized from a 0.2 μm filtered solution in Tris and NaCl.
Reconstitution

Reconstitute at 200 μg/mL in sterile, deionized water.
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.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after reconstitution.

Calculators

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: TIMP-4

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 non-covalent binary complex with the MMP active site through its N-terminal domain. In addition, it binds to the hemopexin-like domain of pro-MMP-2 through its C-terminal domain in a manner similar to TIMP-2 (6). However, unlike TIMP-2, TIMP-4 does not promote pro-MMP-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).

References

  1. Greene, et al. (1996) J. Biol. Chem. 271:30375.
  2. Leco, et al. (1997) FEBS Lett. 401:213.
  3. Geliker, et al. (2001) Oncogene 20:4337.
  4. Groft, et al. (2001) Br. J. Cancer 85:55.
  5. Hurst, et al. (2001) Biochem. Biophys. Res. Comm. 281:166.
  6. Bigg, et al. (1997) J. Biol. Chem. 272:15496.
  7. Hernandez-Barrantes, et al. (2001) Biochem. Biophys. Res. Comm. 281:126.
  8. Amour, et al. (1998) FEBS Lett. 435:39.
  9. Liu, et al. (1997) J. Biol. Chem. 272:20479.

Long Name

Tissue Inhibitors of Metalloproteinases 4

Alternate Names

TIMP4

Entrez Gene IDs

7079 (Human); 110595 (Mouse)

Gene Symbol

TIMP4

UniProt

Q99727

Additional TIMP-4 Products

Product Documents for Recombinant Human TIMP-4 (Sf 21-expressed) Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Human TIMP-4 (Sf 21-expressed) Protein, CF

For research use only

Citations for Recombinant Human TIMP-4 (Sf 21-expressed) Protein, CF

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Protocols

View specific protocols for Recombinant Human TIMP-4 (Sf 21-expressed) Protein, CF (974-TSF):

Materials
  • Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
  • Recombinant human TIMP-4 (rhTIMP-4) (Catalog # 974-TSF)
  • Recombinant Human MMP‑2 (rhMMP‑2) (Catalog # 902-MP)
  • 4-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
  1. Dilute rhMMP-2 to 100 µg/mL with 1 mM APMA in Assay Buffer.
  2. Incubate at 37 ºC for 1 hour (to activate).
  3. Prepare a curve of rhTIMP-4 (MW: 22,400 Da) in Assay Buffer.  Make serial dilutions of: 5000, 2000, 1000, 500, 300, 200, 150, 100, 20, and 2 nM.
  4. After activation, dilute 100 µg/mL rhMMP-2 to 12.5 µg/mL in Assay Buffer.
  5. Mix 16 µL of rhTIMP-4 curve dilutions, 25.6 µL of diluted rhMMP-2, and 118.4 µL of Assay Buffer.  Include a control (in duplicate) containing 134.4 µL of Assay Buffer and 25.6 µL of diluted rhMMP-2.
  6. Incubate reactions for 2 hours at 37 °C.
  7. After incubation, dilute the mixtures five fold in Assay Buffer.
  8. Dilute Substrate to 10 µM in Assay Buffer.
  9. Load 50 µL of the diluted incubated mixtures in a plate, and start the reaction by adding 50 µL of 10 µM Substrate.
  10. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively in kinetic mode for 5 minutes.
  11. Derive the 50% inhibiting concentration (IC50) for rhTIMP-4 by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
  12. 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:  0.020 µg
  • rhTIMP-4:  50, 20, 10, 5, 3, 2, 1.5, 1, 0.2, and 0.02 nM
  • Substrate: 5 µM

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