Recombinant Human TIMP-3 Protein, CF

Catalog # Availability Size / Price Qty
973-TM-010
Product Details
Citations (20)
FAQs
Supplemental Products
Reviews (2)

Recombinant Human TIMP-3 Protein, CF Summary

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 3 nM, under conditions the described conditions.
Recombinant Human TIMP-3 is also an excellent inhibitor of human TACE (ADAM-17), with IC50 values measured in the low nM range.
Source
Mouse myeloma cell line, NS0-derived human TIMP-3 protein
Cys24-Pro211
Accession #
N-terminal Sequence
Analysis
Cys24
Predicted Molecular Mass
22 kDa
SDS-PAGE
26 kDa, reducing conditions

Product Datasheets

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.

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.

Assay Procedure

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
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-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
Entrez Gene IDs
7078 (Human); 21859 (Mouse); 25358 (Rat)
Alternate Names
HSMRK222; K222; K222TA2; metalloproteinase inhibitor 3; MIG-5 protein; Protein MIG-5; pseudoinflammatory); SFD; TIMP metallopeptidase inhibitor 3; TIMP3; TIMP-3; tissue inhibitor of metalloproteinase 3 (Sorsby fundus dystrophy; Tissue inhibitor of metalloproteinases 3

Citations for Recombinant Human TIMP-3 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.

20 Citations: Showing 1 - 10
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  1. Dopamine Receptor Activation Modulates the Integrity of the Perisynaptic Extracellular Matrix at Excitatory Synapses
    Authors: J Mitlöhner, R Kaushik, H Niekisch, A Blondiaux, CE Gee, MFK Happel, E Gundelfing, A Dityatev, R Frischknec, C Seidenbech
    Cells, 2020;9(2):.
    Species: Rat
    Sample Types: Whole Cells
    Applications: Cell Culture
  2. Loss of TIMP3 by promoter methylation of Sp1 binding site promotes oral cancer metastasis
    Authors: CW Su, YC Chang, MH Chien, YH Hsieh, MK Chen, CW Lin, SF Yang
    Cell Death Dis, 2019;10(11):793.
    Species: Human
    Sample Types: Whole Cells
    Applications: Cell Culture
  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;0(0):.
    Species: Human
    Sample Types: Whole Cells
    Applications: Bioassay
  4. Glycosaminoglycans influence enzyme activity of MMP2 and MMP2/TIMP3 complex formation - Insights at cellular and molecular level
    Authors: G Ruiz-Gómez, S Vogel, S Möller, MT Pisabarro, U Hempel
    Sci Rep, 2019;9(1):4905.
    Species: Human
    Sample Types: Cell Culture Supernates
    Applications: Enzyme Assay
  5. TIMP?3 suppresses the proliferation and migration of SMCs from the aortic neck of atherosclerotic AAA in rabbits, via decreased MMP?2 and MMP?9 activity, and reduced TNF?? expression
    Authors: H Zhai, X Qi, Z Li, W Zhang, C Li, L Ji, K Xu, H Zhong
    Mol Med Rep, 2018;18(2):2061-2067.
    Species: Rabbit
    Sample Types: Whole Cells
    Applications: Bioassay
  6. Pericyte ALK5/TIMP3 Axis Contributes to Endothelial Morphogenesis in the Developing Brain
    Authors: JM Dave, T Mirabella, SD Weatherbee, DM Greif
    Dev. Cell, 2018;0(0):.
    Species: Mouse
    Sample Types: In Vivo
    Applications: In Vivo
  7. Characterization of CD200 Ectodomain Shedding
    Authors: KK Wong, F Zhu, I Khatri, Q Huo, DE Spaner, RM Gorczynski
    PLoS ONE, 2016;11(4):e0152073.
    Species: Human
    Sample Types: Whole Cells
    Applications: Bioassay
  8. Cathepsin Protease Controls Copper and Cisplatin Accumulation via Cleavage of the Ctr1 Metal-binding Ecto-domain
    J Biol Chem, 2016;0(0):.
    Species: Mouse
    Sample Types: Whole Cells
    Applications: Bioassay
  9. ADAM17 regulates IL-1 signaling by selectively releasing IL-1 receptor type 2 from the cell surface.
    Authors: Uchikawa S, Yoda M, Tohmonda T, Kanaji A, Matsumoto M, Toyama Y, Horiuchi K
    Cytokine, 2015;71(2):238-45.
    Species: Primate - Chlorocebus pygerythrus (Vervet Monkey)
    Sample Types: Whole Cells
    Applications: Bioassay
  10. IgLON cell adhesion molecules are shed from the cell surface of cortical neurons to promote neuronal growth.
    Authors: Sanz R, Ferraro G, Fournier A
    J Biol Chem, 2015;290(7):4330-42.
  11. ADAM17-mediated shedding of FcgammaRIIIA on human NK cells: identification of the cleavage site and relationship with activation.
    Authors: Lajoie L, Congy-Jolivet N, Bolzec A, Gouilleux-Gruart V, Sicard E, Sung H, Peiretti F, Moreau T, Vie H, Clemenceau B, Thibault G
    J Immunol, 2014;192(2):741-51.
    Species: Human
    Sample Types: Whole Cells
    Applications: Bioassay
  12. ADAM12 is expressed in the tumour vasculature and mediates ectodomain shedding of several membrane-anchored endothelial proteins.
    Authors: Frohlich C, Klitgaard M, Noer J, Kotzsch A, Nehammer C, Kronqvist P, Berthelsen J, Blobel C, Kveiborg M, Albrechtsen R, Wewer U
    Biochem J, 2013;452(1):97-109.
    Species: Human
    Sample Types: Whole Cells
    Applications: Bioassay
  13. Regulated proteolytic processing of Reelin through interplay of tissue plasminogen activator (tPA), ADAMTS-4, ADAMTS-5, and their modulators.
    Authors: Krstic D, Rodriguez M, Knuesel I
    PLoS ONE, 2012;7(10):e47793.
    Species: Human
    Sample Types: Recombinant Protein
    Applications: Enzyme Assay
  14. Insights into ectodomain shedding and processing of protein-tyrosine pseudokinase 7 (PTK7).
    Authors: Golubkov V, Strongin A
    J Biol Chem, 2012;287(50):42009-18.
    Species: Human
    Sample Types: Whole Cells
    Applications: Bioassay
  15. Heparan sulfate-modulated, metalloprotease-mediated sonic hedgehog release from producing cells.
    Authors: Dierker T, Dreier R, Petersen A, Bordych C, Grobe K
    J. Biol. Chem., 2009;284(12):8013-22.
    Species: Chicken
    Sample Types: Whole Cells
    Applications: Bioassay
  16. MMP25 (MT6-MMP) is highly expressed in human colon cancer, promotes tumor growth, and exhibits unique biochemical properties.
    Authors: Sun Q, Weber CR, Sohail A, Bernardo MM, Toth M, Zhao H, Turner JR, Fridman R
    J. Biol. Chem., 2007;282(30):21998-2010.
    Species: Human
    Sample Types: Whole Cells
    Applications: Bioassay
  17. Coregulation of vascular tube stabilization by endothelial cell TIMP-2 and pericyte TIMP-3.
    Authors: Saunders WB, Bohnsack BL, Faske JB, Anthis NJ, Bayless KJ, Hirschi KK, Davis GE
    J. Cell Biol., 2006;175(1):179-91.
    Species: Human
    Sample Types: Whole Cells
    Applications: Bioassay
  18. The shedding activity of ADAM17 is sequestered in lipid rafts.
    Authors: Tellier E, Rebsomen L
    Exp. Cell Res., 2006;312(20):3969-80.
    Species: Human
    Sample Types: Whole Cells
    Applications: Bioassay
  19. Tumor cell invasion of collagen matrices requires coordinate lipid agonist-induced G-protein and membrane-type matrix metalloproteinase-1-dependent signaling.
    Authors: Fisher KE, Pop A, Koh W
    Mol. Cancer, 2006;5(0):69.
    Species: Human
    Sample Types: Whole Cells
    Applications: Bioassay
  20. Depletion of cellular cholesterol and lipid rafts increases shedding of CD30.
    Authors: von Tresckow B, Kallen KJ, von Strandmann EP, Borchmann P, Lange H, Engert A, Hansen HP
    J. Immunol., 2004;172(7):4324-31.
    Species: Human
    Sample Types: Whole Cells
    Applications: Bioassay

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Reviews for Recombinant Human TIMP-3 Protein, CF

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Recombinant Human TIMP-3 Protein, CF
By Anonymous on 05/06/2020
Application: SDS-PAGE Control

Recombinant Human TIMP-3 Protein, CF
By Anonymous on 01/13/2016
Application: In vitro bioactivity in cell culture