Recombinant Human TIMP-2 Protein, CF Summary
Product Specifications
Cys27-Pro220
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.
971-TM
| 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 at ambient temperature. 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, pH 7.5 (TCNB)
- Recombinant Human TIMP-2 (rhTIMP-2) (Catalog # 971-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
- Dilute rhMMP-2 to 100 µg/mL in Assay Buffer with 1 mM of APMA.
- Incubate at 37 °C for 1 hour.
- Prepare a curve of rhTIMP-2 (MW: 21,500 Da). Make the following serial dilutions in Assay Buffer: 2000, 1000, 500, 300, 200, 150, 100, 50, 25, and 2.5 nM.
- Dilute rhMMP-2 to 12.8 µg/mL in Assay Buffer.
- For each inhibition point mix 25 µL of 12.8 µg/mL rhMMP-2, 16 µL of the rhTIMP-2 curve dilution, and 119 µL of Assay Buffer. Include two enzyme controls of 25 µL of 12.8 µg/mL rhMMP-2 and 135 µL Assay Buffer.
- Incubate reaction mixtures at 37 °C for 2 hours.
- Dilute incubated reaction mixtures by 5 fold in Assay Buffer.
- Dilute Substrate to 10 µM in Assay Buffer.
- In a plate load 50 µL of the 5-fold diluted incubated reaction mixtures to wells.
- Start the reaction by adding 50 µL of 10 µM Substrate to all 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 of rhTIMP-2 (IC50) 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).
Per Well:- rhMMP-2: 0.02 µg
- rhTIMP-2 curve: 20, 10, 5, 3, 2, 1.5, 1.0, 0.5, 0.25, 0.025, and 0 nM
- Substrate: 5 µM
Reconstitution Calculator
Background: TIMP-2
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-2 is a non N-glycosylated protein with a molecular mass of 22 kDa produced by a wide range of cell types, which inhibits MMPs non-covalently by the formation of binary complexes. TIMP-2 also has erythroid‑potentiating and cell growth promoting activities.
Citations for Recombinant Human TIMP-2 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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The C-terminal domains of ADAMTS1 contain exosites involved in its proteoglycanase activity
Authors: A Minns, Y Qi, K Yamamoto, K Lee, J Ahnström, S Santamaria
The Journal of Biological Chemistry, 2023-02-21;0(0):103048.
Species: N/A
Sample Types: Recombinant Protein
Applications: Bioassay -
Screening MT1-MMP Activity and Inhibition in Three-Dimensional Tumor Spheroids
Authors: AM Knapinska, G Drotleff, C Chai, D Twohill, A Ernce, D Tokmina-Ro, I Grande, M Rodriguez, B Larson, GB Fields
Biomedicines, 2023-02-15;11(2):.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay -
Vemurafenib induces a noncanonical senescence-associated secretory phenotype in melanoma cells which promotes vemurafenib resistance
Authors: Peng, J;Lin, Z;Chen, W;Ruan, J;Deng, F;Yao, L;Rao, M;Xiong, X;Xu, S;Zhang, X;Liu, X;Sun, X;
Heliyon
Species: Human
Sample Types: Cell Culture Supernates
Applications: Cell Culture -
Post-Translational Regulation And Proteolytic Activity Of The Metalloproteinase Adamts8
Authors: S Santamaria, DR Martin, X Dong, K Yamamoto, SS Apte, J Ahnström
The Journal of Biological Chemistry, 2021-10-21;0(0):101323.
Species: Human
Sample Types: Protein
Applications: Enzyme Activity -
Chemerin acts via CMKLR1 and GPR1 to stimulate migration and invasion of gastric cancer cells: putative role of decreased TIMP-1 and TIMP-2
Authors: JD Kumar, I Aolymat, L Tiszlavicz, Z Reisz, HM Garalla, R Beynon, D Simpson, GJ Dockray, A Varro
Oncotarget, 2019-01-04;10(2):98-112.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay -
Characterization of CD200 Ectodomain Shedding
Authors: KK Wong, F Zhu, I Khatri, Q Huo, DE Spaner, RM Gorczynski
PLoS ONE, 2016-04-25;11(4):e0152073.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay -
ADAM17 regulates IL-1 signaling by selectively releasing IL-1 receptor type 2 from the cell surface.
Authors: Uchikawa S, Yoda M, Tohmonda T, Kanaji A, Matsumoto M, Toyama Y, Horiuchi K
Cytokine, 2014-11-22;71(2):238-45.
Species: Primate - Chlorocebus pygerythrus (Vervet Monkey)
Sample Types: Whole Cells
Applications: Bioassay -
Conditionally replicating adenovirus expressing TIMP2 for ovarian cancer therapy.
Authors: Yang SW, Cody JJ, Rivera AA, Waehler R, Wang M, Kimball KJ, Alvarez RA, Siegal GP, Douglas JT, Ponnazhagan S
Clin. Cancer Res., 2010-11-29;17(3):538-49.
Species: Human
Sample Types:
Applications: Western Blot -
Decorin is a novel antagonistic ligand of the Met receptor.
Authors: Goldoni S, Humphries A, Nystrom A, Sattar S, Owens RT, McQuillan DJ, Ireton K, Iozzo RV
J. Cell Biol., 2009-05-11;185(4):743-54.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay -
MT1-MMP and RECK are involved in human CD34+ progenitor cell retention, egress, and mobilization.
Authors: Vagima Y, Avigdor A, Goichberg P, Shivtiel S, Tesio M, Kalinkovich A, Golan K, Dar A, Kollet O, Petit I, Perl O, Rosenthal E, Resnick I, Hardan I, Gellman YN, Naor D, Nagler A, Lapidot T
J. Clin. Invest., 2009-02-09;119(3):492-503.
Species: Human
Sample Types: Whole Cells
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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This recombinant protein was used as an ELISA standard with MAB9711 and BAF971 as matched pair antibodies.
