Recombinant Human TIMP-3 Protein, CF

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Product Details

Summary
Reactivity HuSpecies Glossary
Applications Inhibition Activity
Format
Carrier-Free

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Recombinant Human TIMP-3 Protein, CF Summary

Details of Functionality
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
Cys24
Protein/Peptide Type
Recombinant Enzymes
Gene
TIMP3
Purity
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

Theoretical MW
22 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
SDS-PAGE
26 kDa, reducing conditions
Publications
Read Publications using
973-TM in the following applications:

Packaging, Storage & Formulations

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.
Buffer
Lyophilized from a 0.2 μm filtered solution in Tris and NaCl.
Purity
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Reconstitution Instructions
Reconstitute at 100 μg/mL in sterile, deionized water.
Assay Procedure
  • 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

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human TIMP-3 Protein, CF

  • 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

Background

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.

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

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Publications for TIMP-3 (973-TM)(15)

We have publications tested in 5 confirmed species: Human, Mouse, Chicken, Primate - Chlorocebus pygerythrus (Vervet Monkey), Rabbit.

We have publications tested in 2 applications: Bioassay, EnzAct.


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Bioassay
(13)
EnzAct
(1)
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Filter By Species
Human
(10)
Mouse
(1)
Chicken
(1)
Primate - Chlorocebus pygerythrus (Vervet Monkey)
(1)
Rabbit
(1)
All Species
Showing Publications 1 - 10 of 15. Show All 15 Publications.
Publications using 973-TM Applications Species
H Zhai, X Qi, Z Li, W Zhang, C Li, L Ji, K Xu, H Zhong 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 Mol Med Rep, 2018;18(2):2061-2067. 2018 [PMID: 29956789] (Bioassay, Rabbit) Bioassay Rabbit
Cathepsin Protease Controls Copper and Cisplatin Accumulation via Cleavage of the Ctr1 Metal-binding Ecto-domain J Biol Chem, 2016;0(0):. 2016 [PMID: 27143361] (Bioassay, Mouse) Bioassay Mouse
KK Wong, F Zhu, I Khatri, Q Huo, DE Spaner, RM Gorczynski Characterization of CD200 Ectodomain Shedding PLoS ONE, 2016;11(4):e0152073. 2016 [PMID: 27111430] (Bioassay, Human) Bioassay Human
Sanz R, Ferraro G, Fournier A IgLON cell adhesion molecules are shed from the cell surface of cortical neurons to promote neuronal growth. J Biol Chem, 2015;290(7):4330-42. 2015 [PMID: 25538237]
Uchikawa S, Yoda M, Tohmonda T, Kanaji A, Matsumoto M, Toyama Y, Horiuchi K ADAM17 regulates IL-1 signaling by selectively releasing IL-1 receptor type 2 from the cell surface. Cytokine, 2015;71(2):238-45. 2015 [PMID: 25461404] (Bioassay, Primate - Chlorocebus pygerythrus (Vervet Monkey)) Bioassay Primate - Chlorocebus pygerythrus (Vervet Monkey)
Lajoie L, Congy-Jolivet N, Bolzec A, Gouilleux-Gruart V, Sicard E, Sung H, Peiretti F, Moreau T, Vie H, Clemenceau B, Thibault G ADAM17-mediated shedding of FcgammaRIIIA on human NK cells: identification of the cleavage site and relationship with activation. J Immunol, 2014;192(2):741-51. 2014 [PMID: 24337742] (Bioassay, Human) Bioassay Human
Frohlich C, Klitgaard M, Noer J, Kotzsch A, Nehammer C, Kronqvist P, Berthelsen J, Blobel C, Kveiborg M, Albrechtsen R, Wewer U ADAM12 is expressed in the tumour vasculature and mediates ectodomain shedding of several membrane-anchored endothelial proteins. Biochem J, 2013;452(1):97-109. 2013 [PMID: 23458101] (Bioassay, Human) Bioassay Human
Golubkov V, Strongin A Insights into ectodomain shedding and processing of protein-tyrosine pseudokinase 7 (PTK7). J Biol Chem, 2012;287(50):42009-18. 2012 [PMID: 23095747] (Bioassay, Human) Bioassay Human
Krstic D, Rodriguez M, Knuesel I Regulated proteolytic processing of Reelin through interplay of tissue plasminogen activator (tPA), ADAMTS-4, ADAMTS-5, and their modulators. PLoS ONE, 2012;7(10):e47793. 2012 [PMID: 23082219] (EnzAct, Human) EnzAct Human
Dierker T, Dreier R, Petersen A, Bordych C, Grobe K Heparan sulfate-modulated, metalloprotease-mediated sonic hedgehog release from producing cells. J. Biol. Chem., 2009;284(12):8013-22. 2009 [PMID: 19176481] (Bioassay, Chicken) Bioassay Chicken
Show All 15 Publications.

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Bioinformatics

Gene Symbol TIMP3
Entrez
Uniprot