Recombinant Human TIMP-2 Protein, CF

Catalog # Availability Size / Price Qty
971-TM-010

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Recombinant Human TIMP-2 Protein, CF Summary

Product Specifications

Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
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 2.2 nM, as measured under the described conditions.
Source
Chinese Hamster Ovary cell line, CHO-derived human TIMP-2 protein
Cys27-Pro220
Accession #
N-terminal Sequence
Analysis
Cys27
Predicted Molecular Mass
21 kDa
SDS-PAGE
22-23 kDa, reducing conditions

Product Datasheets

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971-TM

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after reconstitution.

Assay Procedure

Materials
  • 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
  1. Dilute rhMMP-2 to 100 µg/mL in Assay Buffer with 1 mM of APMA.
  2. Incubate at 37 °C for 1 hour.
  3. 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.
  4. Dilute rhMMP-2 to 12.8 µg/mL in Assay Buffer.
  5. 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.
  6. Incubate reaction mixtures at 37 °C for 2 hours.
  7. Dilute incubated reaction mixtures by 5 fold in Assay Buffer.
  8. Dilute Substrate to 10 µM in Assay Buffer.
  9. In a plate load 50 µL of the 5-fold diluted incubated reaction mixtures to wells.
  10. 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.
  11. Derive the 50% inhibiting concentration of rhTIMP-2 (IC50) 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 (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

Reconstitution Calculator

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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.

Long Name
Tissue Inhibitors of Metalloproteinases 2
Entrez Gene IDs
7077 (Human); 21858 (Mouse); 29543 (Rat)
Alternate Names
CSC-21Ktissue inhibitor of metalloproteinase 2; metalloproteinase inhibitor 2; TIMP metallopeptidase inhibitor 2; TIMP2; TIMP-2; Tissue inhibitor of metalloproteinases 2

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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  1. 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
  2. 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
  3. 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
  4. 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
  5. 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
  6. 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
  7. 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
  8. 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
  9. 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
  10. 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
  11. 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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Recombinant Human TIMP-2 Protein, CF
By Anonymous on 11/16/2017
Application: Immunoassay Standard

This recombinant protein was used as an ELISA standard with MAB9711 and BAF971 as matched pair antibodies.