Mouse TNF RI/TNFRSF1A 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 sTNF RI/TNFRSF1A. 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: TNF RI/TNFRSF1A
TNF RI (TNF receptor 1; TNF R-p55/p60) is a transmembrane receptor for TNF-alpha and Lymphotoxin-alpha/TNF-beta. A soluble decoy form of the receptor is shed by ADAM-17/TACE in response to inflammatory stimulation. TNF RI signaling through NFkB is essential for the development of lymph node germinal centers and Peyer’s patches and for combating intracellular pathogens such as Listeria.
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 TNF RI/TNFRSF1A 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 - 10
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Acute Kidney Injury-Induced Circulating TNFR1/2 Elevations Correlate with Persistent Kidney Injury and Progression to Fibrosis
Authors: Arthanarisami, A;Komaru, Y;Katsouridi, C;Schumacher, J;Verges, DK;Ning, L;Abdelmageed, MM;Herrlich, A;Kefaloyianni, E;
Cells
Species: Mouse
Sample Types: Serum, Urine
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TNFR2 Deficiency Acts in Concert with Gut Microbiota To Precipitate Spontaneous Sex-Biased Central Nervous System Demyelinating Autoimmune Disease.
Authors: Miller P, Bonn M, Franklin C, Ericsson A, McKarns S
J Immunol, 2015-10-16;195(10):4668-84.
Species: Mouse
Sample Types: Cell Culture Supernates
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Dual Inhibition of TNFR1 and IFNAR1 in Imiquimod-Induced Psoriasiform Skin Inflammation in Mice.
Authors: Grine L, Dejager L, Libert C, Vandenbroucke R
J Immunol, 2015-04-24;194(11):5094-102.
Species: Mouse
Sample Types: Tissue Homogenates
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Age-associated aggravation of clinical disease after primary metapneumovirus infection of BALB/c mice.
Authors: Darniot M, Pitoiset C, Petrella T, Aho S, Pothier P, Manoha C
J. Virol., 2009-01-14;83(7):3323-32.
Species: Mouse
Sample Types: BALF
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Elevated urinary VCAM-1, P-selectin, soluble TNF receptor-1, and CXC chemokine ligand 16 in multiple murine lupus strains and human lupus nephritis.
Authors: Wu T, Xie C, Wang HW, Zhou XJ, Schwartz N, Calixto S, Mackay M, Aranow C, Putterman C, Mohan C
J. Immunol., 2007-11-15;179(10):7166-75.
Species: Mouse
Sample Types: Urine
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Tumor necrosis factor-alpha from macrophages enhances LPS-induced clara cell expression of keratinocyte-derived chemokine.
Authors: Elizur A, Adair-Kirk TL, Kelley DG, Griffin GL, deMello DE, Senior RM
Am. J. Respir. Cell Mol. Biol., 2007-08-02;38(1):8-15.
Species: Mouse
Sample Types: Cell Culture Supernates
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Excreted urinary mediators in an animal model of experimental immune nephritis with potential pathogenic significance.
Authors: Wu T, Xie C, Bhaskarabhatla M, Yan M, Leone A, Chen SS, Zhou XJ, Putterman C, Mohan C
Arthritis Rheum., 2007-03-01;56(3):949-59.
Species: Mouse
Sample Types: Urine
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Anti-tumor necrosis factor alpha F(ab')2 antibody fragments protect in murine polymicrobial sepsis: concentration and early intervention are fundamental to the outcome.
Authors: Marquez-Velasco R, Bojalil R, Buelna A, Flores-Guzman F, Estevez-Ramirez J, Laguna J, Hernandez AM, Diaz-Quinonez A, Paniagua-Solis JF
Inflamm. Res., 2006-09-01;55(9):378-84.
Species: Mouse
Sample Types: Serum
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Increased pulmonary responses to acute ozone exposure in obese db/db mice.
Authors: Lu FL, Johnston RA, Flynt L, Theman TA, Terry RD, Schwartzman IN, Lee A, Shore SA
Am. J. Physiol. Lung Cell Mol. Physiol., 2005-12-22;290(5):L856-65.
Species: Mouse
Sample Types: Serum
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Adenoviral infection decreases mortality from lipopolysaccharide-induced liver failure via induction of TNF-alpha tolerance.
Authors: Yarovinsky TO, Powers LS, Butler NS, Bradford MA, Monick MM, Hunninghake GW
J. Immunol., 2003-09-01;171(5):2453-60.
Species: Mouse
Sample Types: Serum
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