Recombinant Rat TIMP-1 Protein, CF

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

Recombinant Rat 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 <3.0 nM, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived rat TIMP-1 protein
  Cys24-Ala217
• Accession #: P30120
• 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: 21.5 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: 32-34 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 Tris and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Reconstitution Instructions: Reconstitute at 500 μg/mL in sterile, deionized water.
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05 % (w/v) Brij-35, pH 7.5 (TCNB)
  • Recombinant Rat TIMP-1 (rrTIMP-1) (Catalog # 580-RT)
  • 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 APMA in Assay Buffer.
  2. Incubate at 37 ºC for 1 hour to activate rhMMP-2.
  3. Dilute activated rhMMP-2 to 12.5 µg/mL in Assay Buffer.
  4. Prepare a curve of rrTIMP-1 (MW: 21,500 Da) in Assay Buffer. Make the following serial dilutions: 5000, 2000, 1000, 500, 300, 200, 150, 100, 20, and 2 nM.
  5. Mix 25.6 µL of 12.5 µg/mL rhMMP-2, 16 µL of rrTIMP-1 serial curve dilutions, and 118.4 µL of Assay Buffer in microtubes. Include two enzyme controls of 25.6 µL of 12.5 µg/mL rhMMP-2 and 134.4 µL Assay Buffer in microtubes.
  6. Incubate reaction mixtures at 37 °C for 2 hours.
  7. Dilute incubated reaction mixtures 5 fold in Assay Buffer.
  8. Dilute Substrate to 10 µM in Assay Buffer.
  9. In a plate load 50 µL of the diluted incubated reaction mixtures to wells, 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. Determine the 50% inhibition concentration (IC₅₀) for rrTIMP-1 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 (pmoles/min/µg) = (Adjusted Vmax* (RFU/min) x Conversion Factor** (pmole/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
  • rrTIMP-1 curve: 50, 20, 10, 5, 3, 2, 1.5, 1, 0.2, 0.02, 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 Rat 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 32‑34 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 (580-RT)(5)

Publications using 580-RT

• RL Sanz, GB Ferraro, MP Girouard, AE Fournier Ectodomain shedding of Limbic System-Associated Membrane Protein (LSAMP) by ADAM Metallopeptidases promotes neurite outgrowth in DRG neurons Sci Rep, 2017-08-11;7(1):7961. 2017-08-11 [PMID: 28801670] (Bioassay, Rat)
• Fjaere E, Andersen C, Myrmel L, Petersen R, Hansen J, Tastesen H, Mandrup-Poulsen T, Brunner N, Kristiansen K, Madsen L, Romer M Tissue Inhibitor Of Matrix Metalloproteinase-1 Is Required for High-Fat Diet-Induced Glucose Intolerance and Hepatic Steatosis in Mice. PLoS ONE, 2015-07-13;10(7):e0132910. 2015-07-13 [PMID: 26168159] (Western Blot, Rat)
• Ramezani-Moghadam M, Wang J, Ho V, Iseli T, Alzahrani B, Xu A, Van der Poorten D, Qiao L, George J, Hebbard L Adiponectin reduces hepatic stellate cell migration by promoting tissue inhibitor of metalloproteinase-1 (TIMP-1) secretion. J Biol Chem, 2015-01-09;290(9):5533-42. 2015-01-09 [PMID: 25575598] (Bioassay, Western Blot, Rat)
• 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, 2014-12-23;290(7):4330-42. 2014-12-23 [PMID: 25538237] (Bioassay, Rat)
• Birukawa N, Murase K, Sato Y, Kosaka A, Yoneda A, Nishita H, Fujita R, Nishimura M, Ninomiya T, Kajiwara K, Miyazaki M, Nakashima Y, Ota S, Murakami Y, Tanaka Y, Minomi K, Tamura Y, Niitsu Y Activated hepatic stellate cells are dependent on self-collagen, cleaved by membrane type 1 matrix metalloproteinase for their growth. J Biol Chem, 2014-05-27;289(29):20209-21. 2014-05-27 [PMID: 24867951] (Bioassay, Rat)

Bioinformatics

• Gene Symbol: Timp1
• Uniprot:
  • P30120()