Biolegend, 767504, Recombinant Human TIMP-4 (carrier free), 25μg

Tissue inhibitors of metalloproteinases (TIMPs) are inhibitors of matrix metalloproteinases (MMPs). This family of inhibitors is consisted of four members, TIMP-1, -2, -3 , and -4.  Their structures are subdivided into an N-terminal and a C-terminal subdomain. A larger N-terminal domain carries the MMP inhibitory activity and a smaller C-terminal domain mediates other non-inhibitory interactions. Human TIMP-4 is the largest of the currently identified TIMPs. It binds to the hemopexin domatin of pro-MMP-2 as TIMP-2 does and inhibits MT1-MMP with high potency. However, TIMP-4 is unable to promote the activation of pro-MMP-2. Expression of TIMP-4 has been observed in heart, kidney, pancreas, colon, testes, brain and adipose tissue. Elevated levels of TIMP-4 have been found in breast, ovary, cervical, prostate, brain, colon, endometrium, and papillary renal tumors. It has been observed that the TIMP-4 expression has been down regulated epigenetically in failing heart.
25μg
Source: Human TIMP-4, amino acids Cys30-Pro224 (Accession # Q99727) with a C-terminal TG-8H-GGQ tag was expressed in CHO cells.
Molecular Mass: The 208 amino acid recombinant protein has a predicted molecular mass of approximately 23.9 kD. The non-reduced and DTT-reduced proteins migrate at 24 and 25 kD by SDS-PAGE, respectively.
Purity: > 95%, as determined by Coomasie stained SDS-PAGE
Formulation: 0.22 µm filtered protein solution is in 20 mM TRIS, 300 mM NaCl, pH 7.5.
Endotoxin Level: Less than 1.0 EU per µg of protein as determined by the LAL method
Concentration: 10 and 25 µg sizes are bottled at 200 µg/mL. 100 µg size and larger sizes are lot-specific and bottled at the concentration indicated on the vial. To obtain lot-specific concentration and expiration, please enter the lot number in our Certificate of Analysis online tool.
Storage & Handling: Unopened vial can be stored between 2°C and 8°C for up to 2 weeks, at -20°C for up to six months, or at -70°C or colder until the expiration date. For maximum results, quick spin vial prior to opening. The protein can be aliquoted and stored at -20°C or colder. Stock solutions can also be prepared at 50 - 100 µg/mL in appropriate sterile buffer, carrier protein such as 0.2 - 1% BSA or HSA can be added when preparing the stock solution. Aliquots can be stored between 2°C and 8°C for up to one week and stored at -20°C or colder for up to 3 months. Avoid repeated freeze/thaw cycles.
Activity: The activity of hTIMP-4 was measured by its ability to inhibit mature hMMP-2. IC50 Value of hTIMP-4 is less than 1.4 nM (34 ng/mL).
Application: Bioassay
Application Notes: Human TIMP-4 Activity assay Human TIMP-4 activity is measured by its ability to inhibit cleavage of a fluorogenic peptide substrate Mca-PLGL(Dpa)AR by activated hMMP-2. The potency of hTIMP-4 is described as IC50 value. MaterialsAssay Buffer: TCNB (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35, pH 7.5)Recombinant Human MMP-2 (Predicted MW: 73.2 kDa)Recombinant human TIMP-4 (Predicted MW: 23.9 kDa)MMP-2 fluorogenic substrate: Mca-PLGL(Dpa)ARP-Aminophenylmercuric acetate (APMA) Activity assay proceduresDilute hMMP-2 in the Assay buffer at 100 µg/mL.Activate hMMP-2 by adding APMA to a final concentration of 1 mM.Incubate the activating mixture for 1 hours at 37 °C.Dilute the activated hMMP-2 to 4 ug/mL (0.4 ng/µL) in Assay Buffer.Make serial dilution of hTIMP-4 in Assay Buffer from 16000 ng/mL to 7.81 ng/mL, and include 0 ng/mL as control.Add into a tube 25 µL of each serially-diluted hTIMP-4 including the control, and then add 25 µL of 4 µg/mL activated hMMP-2.Incubate the mixture at 37 °C for 2 hours.Add 200 µL of Assay Buffer into each tube prepared in step 6 to make an assay mixture.Dilute the substrate in Assay Buffer at 20 µM.Load into a well plate 50 μL of each assay mixture and start the reaction by adding 50 μL of 20 μM Substrate. Include a substrate blank containing 50 μL Assay Buffer and 50 μL of 20 μM Substrate.Read at excitation and emission wavelengths of 320 nm and 405 nm, respectively, in kinetic mode for 5 minutes.Analyze the data with non-linear regression to estimate IC50 value of hTIMP-4 towards hMMP-2. Final assay conditions per wellhMMP-2: 20 ng (200 ng/mL)hTIMP-2: 800, 400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.57, 0.781, 0 ng/mLSubstrate: 10 µMBioLegend carrier-free recombinant proteins provided in liquid format are shipped on blue-ice. Our comparison testing data indicates that when handled and stored as recommended, the liquid format has equal or better stability and shelf-life compared to commercially available lyophilized proteins after reconstitution. Our liquid proteins are verified in-house to maintain activity after shipping on blue ice and are backed by our 100% satisfaction guarantee. If you have any concerns, contact us at [email protected].
Structure: Monomer
Distribution: Brain, adipose tissue, Kidney, and heart; NTERA-2, U-87 MG, and A549 cell line
Function: Regulation of Matrix Metalloproteinase activity
Interaction: MMPs
Ligand/Receptor: MMP-2
Bioactivity: Regulate MMP activities
Biology Area: Angiogenesis, Apoptosis/Tumor Suppressors/Cell Death, Cell Adhesion, Cell Biology
Molecular Family: Enzymes and Regulators
Antigen References: Chaturvedi P, Tyagi S. C. 2016 J Cell Mol Med 20:2089 Liss M. et al. 2009 Am J Pathology 175:940 Melendez-Zajgla J. et al. 2008 Mol Cancer 7:85 Page-McCaw A. et al. 2007 Nat Rev Mol Cell Biol 8:221 Groft et al. 2001 Br J Cancer 85:55. Greene J. et al. 1996 J Biol Chem 271:30375-80
Gene ID: 7079
UniProt: View information about TIMP-4 on UniProt.org
Regulatory Status: RUO
Other Names: Tissue inhibitor of metalloproteinases 4, TIMP-4, TIMP4
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Q: How does the activity of your recombinant proteins compare to competitors?
A: We quality control each and every lot of recombinant protein. Not only do we check its bioactivity, but we also compare it against other commercially available recombinant proteins. We make sure each recombinant protein’s activity is at least as good as or better than the competition’s. In order to provide you with the best possible product, we ensure that our testing process is rigorous and thorough. If you’re curious and eager to make the switch to BioLegend recombinants, contact your sales representative today!
Q: What is the specific activity or ED50 of my recombinant protein?
A: The specific activity range of the protein is indicated on the product datasheets. Because the exact activity values on a per unit basis can largely fluctuate depending on a number of factors, including the nature of the assay, cell density, age of cells/passage number, culture media used, and end user technique, the specific activity is best defined as a range and we guarantee the specific activity of all our lots will be within the range indicated on the datasheet. Please note this only applies to recombinants labeled for use in bioassays. ELISA standard recombinant proteins are not recommended for bioassay usage as they are not tested for these applications.
Q: Have your recombinants been tested for stability?
A: Our testing shows that the recombinant proteins are able to withstand room temperature for a week without losing activity. In addition the recombinant proteins were also found to withstand four cycles of freeze and thaw without losing activity.
Q: Does specific activity of a recombinant protein vary between lots?
A: Specific activity will vary for each lot and for the type of experiment that is done to validate it, but all passed lots will have activity within the established ED50 range for the product and we guarantee that our products will have lot-to-lot consistency. Please conduct an experiment-specific validation to find the optimal ED50 for your system.
Q: How do you convert activity as an ED50 in ng/ml to a specific activity in Units/mg?
A: Use formula Specific activity (Units/mg) = 10^6/ ED50 (ng/mL)