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

Catalog #: 974-TSF
6 Citations Datasheet / COA / SDS
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974-TSF-010
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Recombinant Human TIMP-4 (Sf 21-expressed) Protein, CF Summary

Product Specifications

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.
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.
Source
Spodoptera frugiperda, Sf 21 (baculovirus)-derived human TIMP-4 protein
Cys30-Pro224
Accession #
Q99727
N-terminal Sequence
Analysis
Cys30
Predicted Molecular Mass
22 kDa
SDS-PAGE
23 kDa, reducing conditions

Product Datasheets

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974-TSF

Product Datasheet
COA
SDS

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.

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.

Assay Procedure

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
Reconstitution Calculator

Reconstitution Calculator

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
Entrez Gene IDs
7079 (Human); 110595 (Mouse)
Alternate Names
metalloproteinase inhibitor 4; TIMP metallopeptidase inhibitor 4; TIMP4; TIMP-4; tissue inhibitor of metalloproteinase 4; Tissue inhibitor of metalloproteinases 4

Citations for Recombinant Human TIMP-4 (Sf 21-expressed) 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.

6 Citations: Showing 1 - 6
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  1. The C-terminal domains of ADAMTS1 contain exosites involved in its proteoglycanase activity
    Authors: A Minns, Y Qi, K Yamamoto, K Lee, J Ahnström, S Santamaria
    The Journal of Biological Chemistry, 2023-02-21;0(0):103048.
    Species: N/A
    Sample Types: Recombinant Protein
    Applications: Bioassay
  2. Post-Translational Regulation And Proteolytic Activity Of The Metalloproteinase Adamts8
    Authors: S Santamaria, DR Martin, X Dong, K Yamamoto, SS Apte, J Ahnström
    The Journal of Biological Chemistry, 2021-10-21;0(0):101323.
    Species: Human
    Sample Types: Protein
    Applications: Enzyme Activity
  3. The macrophage-related biomarkers sCD163 and sCD206 are released by different shedding mechanisms
    Authors: MC Nielsen, MN Andersen, N Rittig, S Rødgaard-H, H Grønbaek, SK Moestrup, HJ Møller, A Etzerodt
    J. Leukoc. Biol., 2019-06-26;0(0):.
    Species: Human
    Sample Types: Whole Cells
    Applications: Bioassay
  4. Characterization of CD200 Ectodomain Shedding
    Authors: KK Wong, F Zhu, I Khatri, Q Huo, DE Spaner, RM Gorczynski
    PLoS ONE, 2016-04-25;11(4):e0152073.
    Species: Human
    Sample Types: Whole Cells
    Applications: Bioassay
  5. Tissue Inhibitor of Metalloproteinase-4 Triggers Apoptosis in Cervical Cancer Cells.
    Authors: Lizarraga F, Ceballos-Cancino G, Espinosa M, Vazquez-Santillan K, Maldonado V, Melendez-Zajgla J
    PLoS ONE, 2015-08-20;10(8):e0135929.
    Species: Human
    Sample Types: Whole Cells
    Applications: Bioassay
  6. Insights into ectodomain shedding and processing of protein-tyrosine pseudokinase 7 (PTK7).
    Authors: Golubkov V, Strongin A
    J Biol Chem, 2012-10-24;287(50):42009-18.
    Species: Human
    Sample Types: Whole Cells
    Applications: Bioassay

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