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Recombinant Human TIMP-3 Protein, CF

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Product Details

Summary
Reactivity HuSpecies Glossary
Applications Inhibition Activity
Format
Carrier-Free

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Recombinant Human TIMP-3 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 approximately 3 nM, under conditions the described conditions. 
Source
Mouse myeloma cell line, NS0-derived human TIMP-3 protein
Cys24-Pro211
Accession #
N-terminal Sequence
Cys24
Protein/Peptide Type
Recombinant Enzymes
Gene
TIMP3
Purity
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

Dilutions
  • Inhibition Activity
Theoretical MW
22 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
26 kDa, reducing conditions
Publications
Read Publications using
973-TM in the following applications:

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 visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Reconstitution Instructions
Reconstitute at 100 μg/mL in sterile, deionized water.
Assay Procedure
  • Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35 (v/v), pH 7.5 (TCNB)
  • Recombinant Human TIMP-3 (rhTIMP-3) (Catalog # 973-TM)
  • Recombinant Human MMP‑2 (rhMMP‑2) (Catalog # 902-MP)
  • 4-Aminophenylmercuric acetate (APMA), 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.
  2. Activate 100 µg/mL rhMMP-2 with 1 mM APMA.
  3. Incubate at 37 °C for 1 hour.
  4. Prepare a curve of rhTIMP-3 (MW: 21,700 Da) in Assay Buffer. Make serial dilutions of: 5,000, 2,000, 1,000, 500, 300, 200, 150, 100, 20, and 2 nM.
  5. After activation, dilute 100 µg/mL rhMMP-2 to 12.5 µg/mL in Assay Buffer.
  6. Mix 16 µL of rhTIMP-3 curve dilutions, 25.6 µL of diluted rhMMP-2, and 118.4 µL of Assay Buffer.
  7. Include a control (in duplicate) containing Assay Buffer and the diluted rhMMP-2.
  8. Incubate reactions for 2 hours at 37 °C.
  9. After incubation, dilute the mixtures 5 fold in Assay Buffer.
  10. Dilute Substrate to 10 µM in Assay Buffer.
  11. Load 50 µL of the diluted incubated mixtures in a plate, and start the reaction by adding 50 µL of 10 µM Substrate.
  12. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively in kinetic mode for 5 minutes.
  13. Derive the IC50 value for rhTIMP-3 from the curve.
  14. Calculate specific activity for each point 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: 0.020 µg
  • Substrate: 5 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human TIMP-3 Protein, CF

  • HSMRK222
  • K222
  • K222TA2
  • metalloproteinase inhibitor 3
  • MIG-5 protein
  • Protein MIG-5
  • pseudoinflammatory)
  • SFD
  • TIMP metallopeptidase inhibitor 3
  • TIMP3
  • TIMP-3
  • tissue inhibitor of metalloproteinase 3 (Sorsby fundus dystrophy
  • Tissue inhibitor of metalloproteinases 3

Background

Tissue inhibitors of metalloproteinases (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-3 is a glycoprotein with a molecular mass of 30 kDa produced by a wide range of cell types. TIMP-3 inhibits active MMP-mediated proteolysis by forming a non-covalent binary complex with the MMP active site through its N-terminal domain. In addition, TIMP-3 is the only known member of the TIMP family that is an effective inhibitor of ADAMs such as TACE (1).

TIMP-3 is unique among the TIMPs because of its high affinity for binding to the extracellular matrix (2). Point mutations in the TIMP-3 C-terminal domain have been reported to result in Sorsby's fundus dystrophy, a disease leading to macular degeneration and loss of vision.

  1. Amour, A. et al. (1998) FEBS Lett. 435:39.
  2. Leco, K.J. et al. (1994) J. Biol. Chem. 269:9352.

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Publications for TIMP-3 (973-TM)(27)

We have publications tested in 7 confirmed species: Human, Mouse, Rat, Chicken, N/A, Primate - Chlorocebus pygerythrus (Vervet Monkey), Rabbit.

We have publications tested in 7 applications: Bioassay, Cell Culture, ELISA Detection, Enzyme Activity, Enzyme Assay, In Vivo, In vivo assay.


Filter By Application
Bioassay
(19)
Cell Culture
(2)
ELISA Detection
(1)
Enzyme Activity
(1)
Enzyme Assay
(2)
In Vivo
(2)
In vivo assay
(1)
All Applications
Filter By Species
Human
(18)
Mouse
(3)
Rat
(3)
Chicken
(1)
N/A
(1)
Primate - Chlorocebus pygerythrus (Vervet Monkey)
(1)
Rabbit
(1)
All Species
Showing Publications 1 - 10 of 27. Show All 27 Publications.
Publications using 973-TM Applications Species
Varga, AJ;Nemeth, IB;Kemeny, L;Varga, J;Tiszlavicz, L;Kumar, D;Dodd, S;Simpson, AWM;Buknicz, T;Beynon, R;Simpson, D;Krenacs, T;Dockray, GJ;Varro, A; Elevated Serum Gastrin Is Associated with Melanoma Progression: Putative Role in Increased Migration and Invasion of Melanoma Cells International journal of molecular sciences 2023-11-28 [PMID: 38069171] (Bioassay, Human) Bioassay Human
Abu El-Asrar, AM;Nawaz, MI;Ahmad, A;Dillemans, L;Siddiquei, M;Allegaert, E;Gikandi, PW;De Hertogh, G;Opdenakker, G;Struyf, S; CD40 Ligand-CD40 Interaction Is an Intermediary between Inflammation and Angiogenesis in Proliferative Diabetic Retinopathy International journal of molecular sciences 2023-10-25 [PMID: 37958563] (Bioassay, Human) Bioassay Human
Tonello, R;Silveira Prudente, A;Hoon Lee, S;Faith Cohen, C;Xie, W;Paranjpe, A;Roh, J;Park, CK;Chung, G;Strong, JA;Zhang, JM;Berta, T; Single-Cell Analysis of Dorsal Root Ganglia Reveals Metalloproteinase Signaling in Satellite Glial Cells and Pain Brain, behavior, and immunity 2023-08-07 [PMID: 37557960] (In vivo assay, Mouse) In vivo assay Mouse
A Minns, Y Qi, K Yamamoto, K Lee, J Ahnström, S Santamaria The C-terminal domains of ADAMTS1 contain exosites involved in its proteoglycanase activity The Journal of Biological Chemistry, 2023-02-21;0(0):103048. 2023-02-21 [PMID: 36813235] (Bioassay, N/A) Bioassay N/A
AN Mwaura, MA Riaz, JB Maoga, E Mecha, COA Omwandho, G Scheiner-B, I Meinhold-H, L Konrad Role of Betaglycan in TGF-beta Signaling and Wound Healing in Human Endometriotic Epithelial Cells and in Endometriosis Biology, 2022-03-26;11(4):. 2022-03-26 [PMID: 35453712] (Bioassay, Human) Bioassay Human
AM Abu El-Asr, A Ahmad, MI Nawaz, MM Siddiquei, A De Zutter, L Vanbrabant, PW Gikandi, G Opdenakker, S Struyf Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation Frontiers in Physiology, 2022-01-10;12(0):807747. 2022-01-10 [PMID: 35082694] (Bioassay, In Vivo, Human, Rat) Bioassay, In Vivo Human, Rat
S Santamaria, DR Martin, X Dong, K Yamamoto, SS Apte, J Ahnström Post-Translational Regulation And Proteolytic Activity Of The Metalloproteinase Adamts8 The Journal of Biological Chemistry, 2021-10-21;0(0):101323. 2021-10-21 [PMID: 34687701] (Enzyme Activity, Human) Enzyme Activity Human
J Cassuto, A Folestad, J Göthlin, H Malchau, J Kärrholm Concerted actions by MMPs, ADAMTS and serine proteases during remodeling of the cartilage callus into bone during osseointegration of hip implants Bone Rep, 2020-09-11;13(0):100715. 2020-09-11 [PMID: 32995386] (ELISA Detection, Human) ELISA Detection Human
J Mitlöhner, R Kaushik, H Niekisch, A Blondiaux, CE Gee, MFK Happel, E Gundelfing, A Dityatev, R Frischknec, C Seidenbech Dopamine Receptor Activation Modulates the Integrity of the Perisynaptic Extracellular Matrix at Excitatory Synapses Cells, 2020-01-21;9(2):. 2020-01-21 [PMID: 31972963] (Cell Culture, Rat) Cell Culture Rat
CW Su, YC Chang, MH Chien, YH Hsieh, MK Chen, CW Lin, SF Yang Loss of TIMP3 by promoter methylation of Sp1 binding site promotes oral cancer metastasis Cell Death Dis, 2019-10-17;10(11):793. 2019-10-17 [PMID: 31624299] (Cell Culture, Human) Cell Culture Human
Show All 27 Publications.

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Bioinformatics

Gene Symbol TIMP3
Uniprot