Mouse TIMP-1 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 mouse TIMP-1. 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-1
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 Mouse TIMP-1 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 - 9
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Heat-Inactivated Akkermansia muciniphila Improves Gut Permeability but Does Not Prevent Development of Non-Alcoholic Steatohepatitis in Diet-Induced Obese Ldlr-/-.Leiden Mice
Authors: MC Morrison, E Gart, WV Duyvenvoor, J Snabel, MJ Nielsen, DJ Leeming, A Menke, R Kleemann
International Journal of Molecular Sciences, 2022-02-19;23(4):.
Species: Mouse
Sample Types: Plasma
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Chemerin deficiency regulates adipogenesis is depot different through TIMP1
Authors: CL Huang, LL Xiao, M Xu, J Li, SF Li, CS Zhu, YL Lin, R He, X Li
Genes & diseases, 2020-04-09;8(5):698-708.
Species: Mouse
Sample Types: Cell Culture Supernates
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Oral administration of EPA-rich oil impairs collagen reorganization due to elevated production of IL-10 during skin wound healing in mice
Authors: B Burger, CMC Kühl, T Candreva, RDS Cardoso, JR Silva, BG Castelucci, SR Consonni, HL Fisk, PC Calder, MAR Vinolo, HG Rodrigues
Sci Rep, 2019-06-24;9(1):9119.
Species: Mouse
Sample Types: Tissue Homogenates
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Endotoxemia shifts neutrophils with TIMP-free gelatinase B/ MMP-9 from bone marrow to periphery and induces systematic upregulation of TIMP-1
Authors: J Vandooren, W Swinnen, E Ugarte-Ber, L Boon, D Dorst, E Martens, G Opdenakker
Haematologica, 2017-08-03;0(0):.
Species: Mouse
Sample Types: Tissue Homogenates
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Regulation of adult hematopoiesis by the a disintegrin and metalloproteinase 10 (ADAM10).
Authors: Weber S, Wetzel S, Prox J, Lehmann T, Schneppenheim J, Donners M, Saftig P
Biochem Biophys Res Commun, 2013-11-15;442(3):234-41.
Species: Mouse
Sample Types: Plasma
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Adenosine augments IL-10-induced STAT3 signaling in M2c macrophages.
Authors: Koscso B, Csoka B, Kokai E, Nemeth Z, Pacher P, Virag L, Leibovich S, Hasko G
J Leukoc Biol, 2013-08-06;94(6):1309-15.
Species: Mouse
Sample Types: Cell Culture Supernates
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Tumor microenvironment-derived proteins dominate the plasma proteome response during breast cancer induction and progression.
Authors: Pitteri SJ, Kelly-Spratt KS, Gurley KE
Cancer Res., 2011-06-08;71(15):5090-100.
Species: Mouse
Sample Types: Plasma
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Systemic delivery of TNF-related apoptosis-inducing ligand (TRAIL) elevates levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) and prevents type 1 diabetes in nonobese diabetic mice.
Authors: Kang S, Park EJ, Joe Y, Seo E, Park MK, Seo SY, Chung HY, Yoo YH, Kim DK, Lee HJ
Endocrinology, 2010-11-03;151(12):5638-46.
Species: Mouse
Sample Types: Plasma
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IL-1R1/MyD88 signaling and the inflammasome are essential in pulmonary inflammation and fibrosis in mice.
Authors: Gasse P, Mary C, Guenon I, Noulin N, Charron S, Schnyder-Candrian S, Schnyder B, Akira S, Quesniaux VF, Lagente V, Ryffel B, Couillin I
J. Clin. Invest., 2007-12-01;117(12):3786-99.
Species: Mouse
Sample Types: BALF
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