Recombinant Rat TIMP-1 Protein, CF Summary
Product Specifications
Cys24-Ala217
Analysis
Product Datasheets
580-RT
Carrier Free
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.
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.
580-RT
| Formulation | Lyophilized from a 0.2 μm filtered solution in Tris and NaCl. |
| Reconstitution | Reconstitute at 500 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.
|
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
- Activate rhMMP-2 at 100 µg/mL with 1 mM APMA in Assay Buffer.
- Incubate at 37 ºC for 1 hour to activate rhMMP-2.
- Dilute activated rhMMP-2 to 12.5 µg/mL in Assay Buffer.
- 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.
- 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.
- Incubate reaction mixtures at 37 °C for 2 hours.
- Dilute incubated reaction mixtures 5 fold in Assay Buffer.
- Dilute Substrate to 10 µM in Assay Buffer.
- 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.
- Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
- Determine the 50% inhibition concentration (IC50) for rrTIMP-1 by plotting RFU/min (or specific activity) vs. concentration with 4‑PL fitting.
- 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).
- 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
Background: TIMP-1
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.
Citations for Recombinant Rat TIMP-1 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.
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Citations: Showing 1 - 5
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Ectodomain shedding of Limbic System-Associated Membrane Protein (LSAMP) by ADAM Metallopeptidases promotes neurite outgrowth in DRG neurons
Authors: RL Sanz, GB Ferraro, MP Girouard, AE Fournier
Sci Rep, 2017-08-11;7(1):7961.
Species: Rat
Sample Types: Whole Cells
Applications: Bioassay -
Tissue Inhibitor Of Matrix Metalloproteinase-1 Is Required for High-Fat Diet-Induced Glucose Intolerance and Hepatic Steatosis in Mice.
Authors: Fjaere E, Andersen C, Myrmel L, Petersen R, Hansen J, Tastesen H, Mandrup-Poulsen T, Brunner N, Kristiansen K, Madsen L, Romer M
PLoS ONE, 2015-07-13;10(7):e0132910.
Species: Rat
Sample Types: Tissue Homogenates
Applications: Western Blot -
Adiponectin reduces hepatic stellate cell migration by promoting tissue inhibitor of metalloproteinase-1 (TIMP-1) secretion.
Authors: Ramezani-Moghadam M, Wang J, Ho V, Iseli T, Alzahrani B, Xu A, Van der Poorten D, Qiao L, George J, Hebbard L
J Biol Chem, 2015-01-09;290(9):5533-42.
Species: Rat
Sample Types: Recombinant Protein, Whole Cells
Applications: Bioassay, Western Blot -
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, 2014-12-23;290(7):4330-42.
Species: Rat
Sample Types: Whole Cells
Applications: Bioassay -
Activated hepatic stellate cells are dependent on self-collagen, cleaved by membrane type 1 matrix metalloproteinase for their growth.
Authors: 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
J Biol Chem, 2014-05-27;289(29):20209-21.
Species: Rat
Sample Types: Whole Cells
Applications: Bioassay
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