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Active Tissue Inhibitors Of Metalloproteinase 3 (TIMP3)

SFD; TIMP Metallopeptidase Inhibitor 3; Metallopeptidase Inhibitor 3; Sorsby Fundus Dystrophy; Pseudoinflammatory

ACTIVITY TEST

Tissue Inhibitors Of Metalloproteinase 3 (TIMP3) is an protein belongs to the tissue inhibitor of metalloproteinases family. They are inhibitors of the matrix metalloproteinases. TIMP-3 is the only member of the TIMP family which is found exclusively in the extracellular matrix (ECM). It is regulated in a cell cycle-dependent fashion in certain cell types and may serve as a marker for terminal differentiation. The activity of recombinant mouse TIMP3 was measured by its ability to inhibit rhMMP2 cleavage of a fluorogenic peptide substrate MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH2 in the assay buffer 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5. rhMMP2 was diluted to 100 ug/ml and activated with 1 mM APMA at 37 °C for 1 hour and rmTIMP3 (MW: 23.25 KD) was diluted to different concentrations with the assay buffer. Mix 8 µl of rmTIMP3 curve dilutions, 12.8 µl of activated rhMMP-2, and 59.2 µl of assay buffer, including a control containing assay buffer and the diluted rhMMP-2 and incubate the reactions for 2 hours at 37 °C. Loading 50 µl of the incubated mixtures which were diluted five-fold in assay buffer into empty wells of a plate, and start the reaction by adding 50 µl of 20 µM substrate. Include a substrate blank containing 50 µl of assay buffer and 50 µl of 20 µM substrate. Then read at excitiation and emission wavelengths of 320 nm and 405 nm, respectively, in kinetic mode for 5 minutes. The result was shown in Figure 1 and it was obvious that recombinant mouse TIMP3 significantly decreased rhMMP2 activity. The inhibition IC50 was <26 nM.

USAGE

Reconstitute in 20mM Tris, 150mM NaCl (pH8.0) to a concentration of 0.1-1.0 mg/mL. Do not vortex.

STORAGE

Avoid repeated freeze/thaw cycles. Store at 2-8°C for one month. Aliquot and store at -80°C for 12 months.

STABILITY

The thermal stability is described by the loss rate. The loss rate was determined by accelerated thermal degradation test, that is, incubate the protein at 37°C for 48h, and no obvious degradation and precipitation were observed. The loss rate is less than 5% within the expiration date under appropriate storage condition.

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Magazine Citations
Neuroimmunomodulation Upregulated Expression of Matrix Metalloproteinases and Tissue Inhibitors of Matrix Metalloproteinases in BALB/c Mouse Brain Challenged with Japanese Encephalitis Virus Pubmed: source
Experimental Biology and Medicine Acidic pH conditions mimicking degenerative intervertebral discs impair the survival and biological behavior of human adipose-derived mesenchymal stem cells Rsmjournals: Source
PLoS ONE A Combinatorial Relative Mass Value Evaluation of Endogenous Bioactive Proteins in Three-Dimensional Cultured Nucleus Pulposus Cells of Herniated Intervertebral Discs: Identification of Potential Target Proteins for Gene Therapeutic Approaches Plosone: Source
Physiol. Res. The unwounded skin remodeling in animal models of diabetes types 1 and 2. Pubmed: 24020818
VirusDisease Circulating levels of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases during Japanese encephalitis virus infection Pubmed:26925446
Acta Pharmacol Sin. Pharmacological evidence: a new therapeutic approach to the treatment of chronic heart failure through SUR2B/Kir6.1 channel in endothelial cells. pubmed:27890915
Biochemical genetics Comparison of Selected Protein Levels in Tumour and Surgical Margin in a Group of Patients with Oral Cavity Cancer pubmed:28421310
International Journal of Cancer Circulating and tissue IMP3 levels are correlated with poor survival in renal cell carcinoma Pubmed: 30650187
J. Pers. Med Altered TIMP-3 Levels in the Cerebrospinal Fluid and Plasma of Patients with Alzheimer's Disease Pubmed:35629249
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