Human TIMP-3 DuoSet ELISA Summary
* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.
This DuoSet ELISA Development kit contains the basic components required for the development of sandwich ELISAs to measure natural and recombinant human TIMP-3. The suggested diluent is suitable for the analysis of most cell culture supernate samples. Diluents for complex matrices, such as serum and plasma, should be evaluated prior to use in this DuoSet.
Product Features
- Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
- Development protocols are provided to guide further assay optimization
- Assay can be customized to your specific needs
- Economical alternative to complete kits
Kit Content
- Capture Antibody
- Detection Antibody
- Recombinant Standard
- Streptavidin conjugated to horseradish-peroxidase (Streptavidin-HRP)
Other Reagents Required
DuoSet Ancillary Reagent Kit 2 (5 plates): (Catalog # DY008) containing 96 well microplates, plate sealers, substrate solution, stop solution, plate coating buffer (PBS), wash buffer, and Reagent Diluent Concentrate 2.
The components listed above may be purchased separately:
PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2 - 7.4, 0.2 µm filtered
Wash Buffer: (Catalog # WA126), or 0.05% Tween® 20 in PBS, pH 7.2-7.4
Reagent Diluent: (Catalog # DY995), or 1% BSA in PBS, pH 7.2-7.4, 0.2 µm filtered
Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)
Stop Solution: 2 N H2SO4 (Catalog # DY994)
Microplates: R&D Systems (Catalog # DY990)
Plate Sealers: ELISA Plate Sealers (Catalog # DY992)
Scientific Data
Product Datasheets
Preparation and Storage
Background: TIMP-3
TIMPs-1 through -4 regulate the activity of zinc metalloproteases known as MMPs, ADAMs and ADAMTSs. Structurally, TIMPs contain two domains. The N-terminal domain binds to the active site of mature metalloproteases via a 1:1 non-covalent interaction, blocking access of substrates to the catalytic site. In addition, The C-terminal domain of TIMP-1 and TIMP-2 binds to the hemopexin- like domain of pro-MMP-9 and pro-MMP-2, respectively. The latter binding is essential for the cell surface activation of MMP-2 by MMP-14.
Assay Procedure
GENERAL ELISA PROTOCOL
Plate Preparation
- Dilute the Capture Antibody to the working concentration in PBS without carrier protein. Immediately coat a 96-well microplate with 100 μL per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature.
- Aspirate each well and wash with Wash Buffer, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (400 μL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and blotting it against clean paper towels.
- Block plates by adding 300 μL Reagent Diluent to each well. Incubate at room temperature for a minimum of 1 hour.
- Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
Assay Procedure
- Add 100 μL of sample or standards in Reagent Diluent, or an appropriate diluent, per well. Cover with an adhesive strip and incubate 2 hours at room temperature.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- Add 100 μL of the Detection Antibody, diluted in Reagent Diluent, to each well. Cover with a new adhesive strip and incubate 2 hours at room temperature.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- Add 100 μL of the working dilution of Streptavidin-HRP to each well. Cover the plate and incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
- Repeat the aspiration/wash as in step 2.
- Add 100 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
- Add 50 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
- Determine the optical density of each well immediately, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.
Citations for Human TIMP-3 DuoSet ELISA
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.
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Citations: Showing 1 - 8
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Involvement of the extracellular matrix proteins periostin and tenascin C in nasal polyp remodeling by regulating the expression of MMPs
Authors: K Du, M Wang, N Zhang, P Yu, P Wang, Y Li, X Wang, L Zhang, C Bachert
Clinical and translational allergy, 2021-09-06;11(7):e12059.
Species: Human
Sample Types: Tissue Homogenates
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Genome-wide screens uncover KDM2B as a modifier of protein binding to heparan sulfate
Authors: RJ Weiss, PN Spahn, AWT Chiang, Q Liu, J Li, KM Hamill, S Rother, TM Clausen, MA Hoeksema, BM Timm, K Godula, CK Glass, Y Tor, PLSM Gordts, NE Lewis, JD Esko
Nature Chemical Biology, 2021-04-12;0(0):.
Species: Human
Sample Types: Cell Culture Supernates
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Injectable gelatin microspheres loaded with platelet rich plasma improve wound healing by regulating early inflammation
Authors: S Zhou, L Li, C Chen, Y Chen, L Zhou, FH Zhou, J Dong, L Wang
International Journal of Medical Sciences, 2021-03-03;18(9):1910-1920.
Species: Rat
Sample Types: Cell Culture Supernates
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Exuberant fibroblast activity compromises lung function via ADAMTS4
Authors: DF Boyd, EK Allen, AG Randolph, XJ Guo, Y Weng, CJ Sanders, R Bajrachary, NK Lee, CS Guy, P Vogel, W Guan, Y Li, X Liu, T Novak, MM Newhams, TP Fabrizio, N Wohlgemuth, PM Mourani, PALISI Ped, TN Wight, S Schultz-Ch, SA Cormier, K Shaw-Salib, A Pekosz, RE Rothman, KF Chen, Z Yang, RJ Webby, N Zhong, JC Crawford, PG Thomas
Nature, 2020-10-28;0(0):.
Species: Mouse
Sample Types: BALF
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Glycosaminoglycans influence enzyme activity of MMP2 and MMP2/TIMP3 complex formation - Insights at cellular and molecular level
Authors: G Ruiz-Gómez, S Vogel, S Möller, MT Pisabarro, U Hempel
Sci Rep, 2019-03-20;9(1):4905.
Species: Human
Sample Types: Cell Culture Supernates
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Comparative Analysis of Peptide Composition and Bioactivity of Different Collagen Hydrolysate Batches on Human Osteoarthritic Synoviocytes
Authors: VS Simons, G Lochnit, J Wilhelm, B Ishaque, M Rickert, J Steinmeyer
Sci Rep, 2018-12-07;8(1):17733.
Species: Human
Sample Types: Cell Culture Supernates
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Differential regulation of matrix-metalloproteinases and their tissue inhibitors in patients with aneurysmal subarachnoid hemorrhage.
Authors: Fischer, Marlene, Dietmann, Anelia, Beer, Ronny, Broessner, Gregor, Helbok, Raimund, Pfausler, Bettina, Schmutzhard, Erich, Lackner, Peter
PLoS ONE, 2013-03-28;8(3):e59952.
Species: Human
Sample Types: Serum
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Profile of macrophages in human abdominal aortic aneurysms: a transcriptomic, proteomic, and antibody protein array study.
Authors: Lamblin N, Ratajczak P, Hot D, Dubois E, Chwastyniak M, Beseme O, Drobecq H, Lemoine Y, Koussa M, Amouyel P, Pinet F
J. Proteome Res., 2010-07-02;9(7):3720-9.
Species: Human
Sample Types: Plasma
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We used this kit for the quantification of TIMP-3 in human serum and plasma. Works very well and well-described protocol.
We ran human EDTA-plasma samples at a 1:16 dilution with good precision with this assay.
