Recombinant Mouse TIMP-1 Protein, CF Summary
Product Specifications
Cys25-Arg205
Analysis
Product Datasheets
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.
980-MT
| Formulation | Lyophilized from a 0.2 μm filtered solution in Tris and NaCl. |
| Reconstitution | Reconstitute at 100 μg/mL in sterile 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, and 0.05% Brij-35, pH 7.5. |
| 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% Brij-35 (w/v), pH 7.5 (TCNB)
- Recombinant Mouse TIMP-1 (rmTIMP-1) (Catalog # 980-MT)
- 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
- Dilute rhMMP-2 to 100 µg/mL in Assay Buffer containing 1 mM APMA.
- Incubate at 37 °C for 1 hour (activation).
- Prepare a curve of rmTIMP-1 (MW: 20,200 Da). Make the following serial dilutions in Assay Buffer: 5000, 2000, 1000, 500, 300, 200, 150, 100, 20, and 2 nM.
- Dilute activated rhMMP-2 to 12.5 µg/mL in Assay Buffer.
- Mix 25.6 µL of diluted rhMMP-2, 16 µL of rmTIMP-1 serial curve dilutions, and 118.4 µL of Assay Buffer.
- Include two enzyme controls of 25.6 µL of diluted rhMMP-2 and 134.4 μL Assay Buffer.
- Incubate reaction mixtures at 37 °C for 2 hours.
- Dilute incubated reaction mixtures by a 5-fold dilution 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.
- Start the reaction by adding 50 µL of 10 µM Substrate to wells.
- Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively in kinetic mode for 5 minutes.
- Derive the 50% inhibiting concentration (IC50) for rmTIMP-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 (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).
- rhMMP-2: 0.02 µg
- rmTIMP-1: 50, 20, 10, 5, 3, 2, 1.5, 1, 0.2, and 0.02 nM
- Substrate: 5 µM
Reconstitution Calculator
Background: TIMP-1
Tissue inhibitors of metalloproteinases or TIMPs are a family of homologous proteins that regulate the activity of matrix metalloproteinases (MMPs) (1, 2). There are four known members of the family, TIMP‑1, TIMP-2, TIMP-3 and TIMP-4 that have been found to exhibit multiple functions, including inhibition of active MMPs, proMMP activation, cell growth promotion, matrix binding, inhibition of angiogenesis and the induction of apoptosis. Structurally, TIMPs have two domains, an N‑terminal domain and a C‑terminal domain. Each domain consists of three disulfide-bonded loops. TIMP-1 is a glycoprotein produced by a wide range of cell types. Through its N‑terminal domain, TIMP-1 inhibits active MMPs by forming a non-covalent binary complex with the MMP active site. The C-terminal domain of TIMP-1 interacts with the C-terminal domain of proMMP-9, which may play a role in regulating proMMP-9 activation.
- Murphy, G. and F. Willenbrock (1995) Methods Enzymol. 248:496.
- Brew, K. et al. (2000) Biochim. Biophys. Acta 1477:267.
Citations for Recombinant Mouse 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.
11
Citations: Showing 1 - 10
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Inflammatory and alternatively activated macrophages independently induce metaplasia but cooperatively drive pancreatic precancerous lesion growth
Authors: Geou-Yarh Liou, Alicia K. Fleming Martinez, Heike R. Döppler, Ligia I. Bastea, Peter Storz
iScience
Species: Mouse
Sample Types: Whole Cells
Applications: Bioassay -
Astrocytic TIMP-1 regulates production of Anastellin, a novel inhibitor of oligodendrocyte differentiation and FTY720 responses
Authors: PA Sutter, CM Willis, A Menoret, AM Nicaise, A Sacino, AH Sikkema, E Jellison, KK Win, DK Han, W Church, W Baron, AT Vella, SJ Crocker
bioRxiv : the preprint server for biology, 2023-02-18;0(0):.
Species: Transgenic Mouse
Sample Types: Whole Cells
Applications: Bioassay -
TGF&beta signaling curbs cell fusion and muscle regeneration
Authors: F Girardi, A Taleb, M Ebrahimi, A Datye, DG Gamage, C Peccate, L Giordani, DP Millay, PM Gilbert, B Cadot, F Le Grand
Nature Communications, 2021-02-02;12(1):750.
Species: Mouse
Sample Types: Whole Cells
Applications: Bioassay -
Tissue Inhibitor of Metalloproteinase-1 Promotes Polymorphonuclear Neutrophil (PMN) Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces
Authors: X Wang, J Rojas-Quin, J Wilder, Y Tesfaigzi, D Zhang, CA Owen
J. Immunol., 2019-04-24;0(0):.
Species: Transgenic Mouse
Sample Types: Whole Cells
Applications: Bioassay -
Epiregulin and EGFR interactions are involved in pain processing
Authors: LJ Martin, SB Smith, A Khoutorsky, CA Magnussen, A Samoshkin, RE Sorge, C Cho, N Yosefpour, S Sivaselvac, S Tohyama, T Cole, TM Khuong, E Mir, DG Gibson, JS Wieskopf, SG Sotocinal, JS Austin, CB Meloto, JH Gitt, C Gkogkas, N Sonenberg, JD Greenspan, RB Fillingim, R Ohrbach, GD Slade, C Knott, R Dubner, AG Nackley, A Ribeiro-da, GG Neely, W Maixner, DV Zaykin, JS Mogil, L Diatchenko
J. Clin. Invest., 2017-08-07;0(0):.
Species: Mouse
Sample Types: In Vivo
Applications: In Vivo -
Inhibition of lipopolysaccharide-induced osteoclast formation and bone resorption in vitro and in vivo by cysteine proteinase inhibitors
Authors: F Strålberg, A Kassem, F Kasprzykow, M Abrahamson, A Grubb, C Lindholm, UH Lerner
J. Leukoc. Biol, 2017-02-14;0(0):.
Species: Mouse
Sample Types: Whole Cells
Applications: Bioassay -
Low-density lipoprotein receptor-related protein-1 mediates endocytic clearance of tissue inhibitor of metalloproteinases-1 and promotes its cytokine-like activities.
Authors: Thevenard J, Verzeaux L, Devy J, Etique N, Jeanne A, Schneider C, Hachet C, Ferracci G, David M, Martiny L, Charpentier E, Khrestchatisky M, Rivera S, Dedieu S, Emonard H
PLoS ONE, 2014-07-30;9(7):e103839.
Species: Hamster, Mouse
Sample Types: Whole Cells
Applications: Bioassay -
Gene expression profiling of early hepatic stellate cell activation reveals a role for Igfbp3 in cell migration.
Authors: Mannaerts, Inge, Schroyen, Ben, Verhulst, Stefaan, Van Lommel, Leentje, Schuit, Frans, Nyssen, Marc, van Grunsven, Leo A
PLoS ONE, 2013-12-17;8(12):e84071.
Species: Mouse
Sample Types: Whole Cells
Applications: Bioassay -
Cysteine proteinase inhibitors regulate human and mouse osteoclastogenesis by interfering with RANK signaling.
Authors: Stralberg F, Henning P, Gjertsson I, Kindlund B, Souza P, Persson E, Abrahamson M, Kasprzykowski F, Grubb A, Lerner U
FASEB J, 2013-04-09;27(7):2687-701.
Species: Mouse
Sample Types: Whole Cells
Applications: Bioassay -
Microbial exposure early in life regulates airway inflammation in mice after infection with Streptococcus pneumoniae with enhancement of local resistance.
Authors: Yasuda Y, Matsumura Y, Kasahara K, Ouji N, Sugiura S, Mikasa K, Kita E
Am. J. Physiol. Lung Cell Mol. Physiol., 2009-09-25;298(1):L67-78.
Species: Mouse
Sample Types: Tissue Homogenates
Applications: Bioassay -
Matrix metalloproteinase-2 (MMP-2) regulates astrocyte motility in connection with the actin cytoskeleton and integrins.
Authors: Ogier C, Bernard A, Chollet AM, LE Diguardher T, Hanessian S, Charton G, Khrestchatisky M, Rivera S
Glia, 2006-09-01;54(4):272-84.
Species: Mouse
Sample Types: Whole Cells
Applications: Bioassay
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