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Active Tissue Inhibitors Of Metalloproteinase 2 (TIMP2)

CSC-21K; TIMP Metallopeptidase Inhibitor 2

ACTIVITY TEST

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 erythroidpotentiating and cell growth promoting activities. The activity of recombinant bovine TIMP2 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 rbTIMP2 (MW: 51.75 KD) was diluted to different concentrations with the assay buffer. Mix 8 µl of rhTIMP2 curve dilutions, 12.8 µl of activated rbMMP-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 bovine TIMP2 significantly decreased rhMMP2 activity. The inhibition IC50 was <30 nM.

USAGE

Reconstitute in 10mM PBS (pH7.4) 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
The Journal of Nutrition Oral Administration of 3,3′-Diindolylmethane Inhibits Lung Metastasis of 4T1 Murine Mammary Carcinoma Cells in BALB/c Mice Nutrition&Metabolism: source
Breast Cancer Research and Treatment Oral administration of benzyl-isothiocyanate inhibits solid tumor growth and lung metastasis of 4T1 murine mammary carcinoma cells in BALB/c mice SpringerLink: f3w831267267p277
Moleculer Vision Bone morphogenetic protein-2: a potential regulator in scleral remodeling PubMed: PMC2605409
Experimental & Translational Stroke Medicine Early outcome and blood-brain barrier integrity after co-administered thrombolysis and hyperbaric oxygenation in experimental stroke PubMed: 21679435
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
Polski Przeglad Chirurgiczny Concentration of gelatinases and their tissue inhibitors in pancreatic inflammatory and neoplastic tumors and their influence on the early postoperative course Pubmed: 23585206
Thromb Haemost. Analysis of the expression of nine secreted matrix metalloproteinases and their endogenous inhibitors in the brain of mice subjected to ischaemic stroke Pubmed:24671655
Pediatric Anesthesia? Impact of 6% hydroxyethyl starch 130/0.42 and 5% gelatin on renal function in a pediatric animal model Pubmed:24916275
PLOS ONE The Complementary Effects of Atorvastatin and Exercise Treatment on the Composition and Stability of the Atherosclerotic Plaques in ApoE Knockout Mice Pubmed:25264981
Brain, Behavior, and Immunity Plasma inflammatory biomarkers for Huntington’s disease patients and mouse model Pubmed:25266150
Mediators of Inflammation Metalloproteinases and Their Tissue Inhibitors in Comparison between Different Chronic Pneumopathies in the Horse Journals: Mi
Brain, Behavior, and Immunity Plasma inflammatory biomarkers for Huntington’s disease patients and mouse model PubMed: 25266150
physiological research The skin remodeling in type 1 diabetes and insulin resistance animal models PubMed: 26047379
Physiol Res The skin remodeling in type 1 diabetes and insulin resistance animal models. PubMed: 26047379
J Periodontal Implant Sci Assessment of MMP-1, MMP-8 and TIMP-2 in experimental periodontitis treated with kaempferol pmc:PMC4848383
Int Wound J. From varices to venous ulceration: the story of chronic venous disease described by metalloproteinases. Pubmed:26991748
BMC Veterinary Research Metalloproteinases and their inhibitors are influenced by inhalative glucocorticoid therapy incombination with environmental dust reduction in equine recurrent airway obstruction. pubmed:27938355
Journal of Applied Animal Research Hypoxia enhances secretion of matrix metalloproteinases in bovine dermal fibroblasts:an in vitro approach to bovine digital dermatitis 10.108:09712119.2017.1335208
Cellular Physiology and Biochemistry The MiR-495/Annexin A3/P53 Axis Inhibits the Invasion and EMT of Colorectal Cancer Cells pubmed:29224019
International Wound Journal From varices to venous ulceration: the story of chronic venous disease described by metalloproteinases pubmed:26991748
Journal of Applied Animal Research  Hypoxia enhances secretion of matrix metalloproteinases in bovine dermal fibroblasts: an approach to bovine digital dermatitis 10.1080:09712119.2017.1335208
Mediators of Inflammation Metalloproteinases and their Inhibitors under the Course of Immunostimulation by CPG-ODN and Specific Antigen Inhalation in Equine Asthma
Journal of endovascular therapy: an official journal of the International Society of Endovascular Specialists Correlation of Baseline Plasma and Inguinal Connective Tissue Metalloproteinases and Their Inhibitors With Late High-Pressure Endoleak After Endovascular … Pubmed: 31464166
Scientific Reports Matrix metallopeptidase expression and modulation by transforming growth factor-β1 in equine endometrosis Pubmed: 31980722
MEDIATORS OF INFLAMMATION MMP-9 Concentration in Peritoneal Fluid Is a Valuable Biomarker Associated with Endotoxemia in Equine Colic 33488296
Calcium Binding Protein S100A16 Expedites Proliferation, Invasion and EMT Process in Gastric Cancer 34650982
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