Recombinant Human TIMP-3 Protein, CF

R&D Systems | Catalog # 973-TM

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Key Product Details

  • R&D Systems NS0-derived Recombinant Human TIMP-3 Protein (973-TM)
  • 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

P35625

Applications

Inhibition Activity
View all TIMP-3 Proteins and Enzymes »

Product Specifications

Source

Mouse myeloma cell line, NS0-derived human TIMP-3 protein
Cys24-Pro211

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

Cys24

Predicted Molecular Mass

22 kDa

SDS-PAGE

26 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 3 nM, under conditions the described conditions. 

Reviewed Applications

Read 2 reviews rated 4.5 using 973-TM in the following applications:

Formulation, Preparation, and Storage

973-TM
Formulation Lyophilized from a 0.2 μm filtered solution in Tris and NaCl.
Reconstitution

Reconstitute at 100 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-3

Tissue inhibitors of metalloproteinases (TIMPs) are a family of proteins that regulate the activation and proteolytic activity of the zinc enzymes known as matrix metalloproteinases (MMPs). There are four members of the family, TIMP-1, TIMP-2, TIMP-3 and TIMP-4. TIMP-3 is a glycoprotein with a molecular mass of 30 kDa produced by a wide range of cell types. TIMP-3 inhibits active MMP-mediated proteolysis by forming a non-covalent binary complex with the MMP active site through its N-terminal domain. In addition, TIMP-3 is the only known member of the TIMP family that is an effective inhibitor of ADAMs such as TACE (1).

TIMP-3 is unique among the TIMPs because of its high affinity for binding to the extracellular matrix (2). Point mutations in the TIMP-3 C-terminal domain have been reported to result in Sorsby's fundus dystrophy, a disease leading to macular degeneration and loss of vision.

References

  1. Amour, A. et al. (1998) FEBS Lett. 435:39.
  2. Leco, K.J. et al. (1994) J. Biol. Chem. 269:9352.

Long Name

Tissue Inhibitors of Metalloproteinases 3

Alternate Names

TIMP3

Entrez Gene IDs

7078 (Human); 21859 (Mouse); 25358 (Rat)

Gene Symbol

TIMP3

UniProt

P35625

Additional TIMP-3 Products

Product Documents for Recombinant Human TIMP-3 Protein, CF

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot or batch number in the search box below.

Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Human TIMP-3 Protein, CF

For research use only

Citations for Recombinant Human TIMP-3 Protein, CF

Customer Reviews for Recombinant Human TIMP-3 Protein, CF (2)

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Protocols

View specific protocols for Recombinant Human TIMP-3 Protein, CF (973-TM):

Materials
  • Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35 (v/v), pH 7.5 (TCNB)
  • Recombinant Human TIMP-3 (rhTIMP-3) (Catalog # 973-TM)
  • Recombinant Human MMP‑2 (rhMMP‑2) (Catalog # 902-MP)
  • 4-Aminophenylmercuric acetate (APMA), 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 in Assay Buffer.
  2. Activate 100 µg/mL rhMMP-2 with 1 mM APMA.
  3. Incubate at 37 °C for 1 hour.
  4. Prepare a curve of rhTIMP-3 (MW: 21,700 Da) in Assay Buffer. Make serial dilutions of: 5,000, 2,000, 1,000, 500, 300, 200, 150, 100, 20, and 2 nM.
  5. After activation, dilute 100 µg/mL rhMMP-2 to 12.5 µg/mL in Assay Buffer.
  6. Mix 16 µL of rhTIMP-3 curve dilutions, 25.6 µL of diluted rhMMP-2, and 118.4 µL of Assay Buffer.
  7. Include a control (in duplicate) containing Assay Buffer and the diluted rhMMP-2.
  8. Incubate reactions for 2 hours at 37 °C.
  9. After incubation, dilute the mixtures 5 fold in Assay Buffer.
  10. Dilute Substrate to 10 µM in Assay Buffer.
  11. Load 50 µL of the diluted incubated mixtures in a plate, and start the reaction by adding 50 µL of 10 µM Substrate.
  12. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively in kinetic mode for 5 minutes.
  13. Derive the IC50 value for rhTIMP-3 from the curve.
  14. Calculate specific activity for each point 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
  • Substrate: 5 µM

FAQs

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