Recombinant Human TIMP-1 Protein, CF

• Reactivity: Hu
• Applications: Inhibition Activity
• Format: Carrier-Free

Recombinant Human TIMP-1 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 2.5 nM as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human TIMP-1 protein
  Cys24-Ala207
• Accession #: Q6FGX5
• N-terminal Sequence: Cys24
• Protein/Peptide Type: Recombinant Enzymes
• Gene: TIMP1
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Inhibition Activity
• Theoretical MW: 21 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: 28-30 kDa, reducing conditions

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 Sodium Acetate and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• 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 (w/v), pH 7.5 (TCNB)
  • Recombinant Human TIMP-1 (rhTIMP-1) (Catalog # 970-TM)
  • Recombinant Human MMP‑2 (rhMMP‑2) (Catalog # 902-MP)
  • p-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. Activate rhMMP-2 at 100 µg/mL with 1 mM of APMA at final concentration respectively, in Assay Buffer.
  2. Incubate activation of rhMMP-2 at 37°C for 1 hour.
  3. Prepare a curve of rhTIMP-1 (MW: 20,695 Da). Make the following serial dilutions in Assay Buffer: 2000 nM, 1,000 nM, 500 nM, 300 nM, 200 nM, 150 nM, 100 nM, 20 nM, and 2 nM.
  4. Dilute activated 100 µg/mL rhMMP-2 to 12.8 µg/mL in Assay Buffer.
  5. Mix 25 µL of 12.8 µg/mL rhMMP-2, 16 µL of rhTIMP-1 serial curve dilutions, and 119 µL of Assay Buffer in micro-tubes.
  6. Include two enzyme controls of 25 µL of 12.8 µg/mL rhMMP-2 and 135 mL Assay Buffer in micro-tubes.
  7. Incubate reaction mixtures at 37 °C for 2 hours.
  8. Dilute incubated reaction mixtures by a 5-fold dilution in Assay Buffer.
  9. Dilute Substrate to 10 µM in Assay Buffer.
  10. In a plate load 50 µL of 5-fold diluted incubated reaction mixtures to wells.
  11. Start the reaction by adding 50 µL of 10 µM Substrate to wells.
  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 of rhTIMP-1 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: 0.02 µg
  • rhTIMP-1 curve: 20 nM, 10 nM, 5 nM, 3 nM, 2 nM, 1.5 nM, 1.0 nM, 0.2 nM, 0.02 nM, and 0 nM
  • 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-1 Protein, CF

• CLGI
• Collagenase inhibitor
• collagenase inhibitor)
• EPATIMP-1
• EPO
• erythroid potentiating activity
• Erythroid-potentiating activity
• Fibroblast collagenase inhibitor
• FLJ90373
• HCI
• metalloproteinase inhibitor 1
• TIMP metallopeptidase inhibitor 1
• TIMP1
• TIMP-1
• TIMPtissue inhibitor of metalloproteinase 1 (erythroid potentiating activity
• Tissue inhibitor of metalloproteinases 1

Background

Tissue inhibitors of metalloproteinases or 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-1 is a glycoprotein with a molecular mass of 28 kDa produced by a wide range of cell types. TIMP-1 inhibits active MMP-mediated proteolysis by forming an N-terminal, non-covalent binary complex with the MMP active site. TIMP-1 also associates C-terminally with Pro-MMP-9 in a complex which may play a role in regulating activation. Independent of MMPs, TIMP-1 has been shown to have a role in tissue homeostasis.

Publications for TIMP-1 (970-TM)(20)

Publications using 970-TM

• A Minns, Y Qi, K Yamamoto, K Lee, J Ahnström, S Santamaria The C-terminal domains of ADAMTS1 contain exosites involved in its proteoglycanase activity The Journal of Biological Chemistry, 2023-02-21;0(0):103048. 2023-02-21 [PMID: 36813235] (Bioassay, N/A)
• SK Chintala, J Pan, S Satapathy, R Condruti, Z Hao, PW Liu, CF O'Conner, JT Barr, MR Wilson, S Jeong, ME Fini Recombinant Human Clusterin Seals Damage to the Ocular Surface Barrier in a Mouse Model of Ophthalmic Preservative-Induced Epitheliopathy International Journal of Molecular Sciences, 2023-01-04;24(2):. 2023-01-04 [PMID: 36674497] (In Vivo, Mouse)
• QC Larrouture, AP Cribbs, SR Rao, M Philpott, SJ Snelling, HJ Knowles Loss of mutual protection between human osteoclasts and chondrocytes in damaged joints initiates osteoclast-mediated cartilage degradation by MMPs Scientific Reports, 2021-11-22;11(1):22708. 2021-11-22 [PMID: 34811438] (Bioassay, Human)
• S Santamaria, DR Martin, X Dong, K Yamamoto, SS Apte, J Ahnström Post-Translational Regulation And Proteolytic Activity Of The Metalloproteinase Adamts8 The Journal of Biological Chemistry, 2021-10-21;0(0):101323. 2021-10-21 [PMID: 34687701] (Enzyme Activity, Human)
• Alee Choi, Sang Eon Park, Jang Bin Jeong, Suk-joo Choi, Soo-young Oh, Gyu Ha Ryu et al. Anti-Fibrotic Effect of Human Wharton’s Jelly-Derived Mesenchymal Stem Cells on Skeletal Muscle Cells, Mediated by Secretion of MMP-1 International Journal of Molecular Sciences 2020-08-29 [PMID: 32872523] (Bioassay, Mouse)
• U Mirastschi, B Lupše, K Maedler, B Sarma, A Radtke, G Belge, M Dorsch, D Wedekind, LJ McCawley, G Boehm, U Zier, K Yamamoto, S Kelm, MS Ågren Matrix Metalloproteinase-3 is Key Effector of TNF-alpha-Induced Collagen Degradation in Skin Int J Mol Sci, 2019-10-22;20(20):. 2019-10-22 [PMID: 31652545] (Bioassay, Mouse)
• JD Kumar, I Aolymat, L Tiszlavicz, Z Reisz, HM Garalla, R Beynon, D Simpson, GJ Dockray, A Varro Chemerin acts via CMKLR1 and GPR1 to stimulate migration and invasion of gastric cancer cells: putative role of decreased TIMP-1 and TIMP-2 Oncotarget, 2019-01-04;10(2):98-112. 2019-01-04 [PMID: 30719206] (Bioassay, Human)
• KK Wong, F Zhu, I Khatri, Q Huo, DE Spaner, RM Gorczynski Characterization of CD200 Ectodomain Shedding PLoS ONE, 2016-04-25;11(4):e0152073. 2016-04-25 [PMID: 27111430] (Bioassay, Human)
• 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, 2014-11-22;71(2):238-45. 2014-11-22 [PMID: 25461404] (Bioassay, Primate - Chlorocebus pygerythrus (Vervet Monkey))
• Agren M, Schnabel R, Christensen L, Mirastschijski U Tumor necrosis factor-alpha-accelerated degradation of type I collagen in human skin is associated with elevated matrix metalloproteinase (MMP)-1 and MMP-3 ex vivo. Eur J Cell Biol, 2014-10-23;94(1):12-21. 2014-10-23 [PMID: 25457675] (Tissue Culture, Human)
• Vandooren J, Born B, Solomonov I, Zajac E, Saldova R, Senske M, Ugarte-Berzal E, Martens E, Van den Steen P, Van Damme J, Garcia-Pardo A, Froeyen M, Deryugina E, Quigley J, Moestrup S, Rudd P, Sagi I, Opdenakker G Circular trimers of gelatinase B/matrix metalloproteinase-9 constitute a distinct population of functional enzyme molecules differentially regulated by tissue inhibitor of metalloproteinases-1. Biochem J, 2015-01-15;465(2):259-70. 2015-01-15 [PMID: 25360794] (Bioassay, Mouse)
• Vinther L, Lademann U, Andersen E, Hojrup P, Thaysen-Andersen M, Krogh B, Viuff B, Brunner N, Stenvang J, Moreira J Purification and characterization of bioactive his6-tagged recombinant human tissue inhibitor of metalloproteinases-1 (TIMP-1) protein expressed at high yields in mammalian cells. Protein Expr Purif, 2014-07-03;101(0):157-64. 2014-07-03 [PMID: 24998777] (Enzyme Assay, Human)
• Tinnes , Stefanie, Ringwald , Julia, Haas , Carola A TIMP-1 inhibits the proteolytic processing of Reelin in experimental epilepsy. FASEB J, 2013-03-14;27(7):2542-52. 2013-03-14 [PMID: 23493620] (Bioassay, Rat)
• Meissburger B, Stachorski L, Roder E, Rudofsky G, Wolfrum C Tissue inhibitor of matrix metalloproteinase 1 (TIMP1) controls adipogenesis in obesity in mice and in humans. Diabetologia, 2011-03-25;54(6):1468-79. 2011-03-25 [PMID: 21437772] (Enzyme Inhibition, Human, Mouse)
• Schipper HS, De Jager W, van Dijk ME, Meerding J, Zelissen PM, Adan RA, Prakken BJ, Kalkhoven E A multiplex immunoassay for human adipokine profiling. Clin. Chem., 2010-06-08;56(0):1320. 2010-06-08 [PMID: 20530731] (ELISA (Standard))
• Goldoni S, Humphries A, Nystrom A, Sattar S, Owens RT, McQuillan DJ, Ireton K, Iozzo RV Decorin is a novel antagonistic ligand of the Met receptor. J. Cell Biol., 2009-05-11;185(4):743-54. 2009-05-11 [PMID: 19433454] (Bioassay, Human)
• Nicol GR, Han M, Kim J, Birse CE, Brand E, Nguyen A, Mesri M, FitzHugh W, Kaminker P, Moore PA, Ruben SM, He T Use of an immunoaffinity-mass spectrometry-based approach for the quantification of protein biomarkers from serum samples of lung cancer patients. Mol. Cell Proteomics, 2008-04-03;7(10):1974-82. 2008-04-03 [PMID: 18388126]
• Anghelina M, Krishnan P, Moldovan L, Moldovan NI Monocytes/macrophages cooperate with progenitor cells during neovascularization and tissue repair: conversion of cell columns into fibrovascular bundles. Am. J. Pathol., 2006-02-01;168(2):529-41. 2006-02-01 [PMID: 16436667] (Bioassay, Mouse)
• Djafarzadeh R, Noessner E, Engelmann H GPI-anchored TIMP-1 treatment renders renal cell carcinoma sensitive to FAS-meditated killing. Oncogene, 2006;25(10):1496-508. 2006 [PMID: 16261161] (ELISA Standard, N/A)
• von Tresckow B, Kallen KJ, von Strandmann EP, Borchmann P, Lange H, Engert A, Hansen HP Depletion of cellular cholesterol and lipid rafts increases shedding of CD30. J. Immunol., 2004-04-01;172(7):4324-31. 2004-04-01 [PMID: 15034047] (Bioassay, Human)

Bioinformatics

• Gene Symbol: TIMP1
• Uniprot:
  • Q6FGX5()