Mouse RAGE 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.
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
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 equivalent
Reagent Diluent: 1% BSA in PBS, pH 7.2-7.4, 0.2 m filtered (R&D Systems Catalog # DY995). Quality of BSA is critical (see Technical Hints).
Blocking Buffer: 1% BSA in PBS, pH 7.2-7.4, 0.2 m filtered (R&D Systems Catalog # DY995). Quality of BSA is critical (see Technical Hints).
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), or equivalent
Plate Sealers: ELISA Plate Sealers (Catalog # DY992), or equivalent
Scientific Data
Product Datasheets
Preparation and Storage
Background: RAGE/AGER
RAGE (Receptor for Advanced Glycation End product) is a transmembrane glycoprotein that binds advanced glycation end products (AGEs), beta-amyloid peptides, HMGB1/Amphoterin, and several S100 family proteins. AGEs are adducts formed by the non-enzymatic glycation and oxidation of proteins and lipids. A soluble form can also be generated by MMP-mediated shedding. RAGE is expressed in the CNS during development as well as in adult endothelial cells, smooth muscle cells, pericytes, monocytes, and neurons. It is locally upregulated in vascular inflammation (e.g. diabetes, atherosclerosis, vascular injury, Alzheimer’s disease). At these sites, RAGE binding to S100A1, EN-RAGE/S100A12, or S100B induces inflammatory immune cell adhesion and infiltration as well as vascular smooth muscle proliferation, neointimal expansion, atherosclerotic plaque development, and transport of A-beta into the cerebrospinal fluid. In cancer, RAGE binding to HMGB1, S100A8, or S100A9 promotes tumor growth and metastasis in addition to inflammatory cell infiltration.
To view our complete solutions for RAGE research, visit bio-techne.com.
Assay Procedure
GENERAL ELISA PROTOCOL
Plate Preparation
- Dilute the Capture Antibody (to the working concentration stated in the product datasheet ) 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 each well of the microplate as recommended in the product datasheet. Incubate at room temperature for a minimum of 1 hour.
Note: The recommended Reagent Diluent typically contains 1% BSA. Some DuoSet Development Kits require alternative blocking agents, or for plates to be blocked overnight with a higher percentage of BSA, please see the product datasheet for details.
- Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
PRECAUTION
The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face and clothing protection when using this material.
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 (as recommended in the product datasheet), 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 RAGE 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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Severe but not moderate hyperoxia of newborn mice causes an emphysematous lung phenotype in adulthood without persisting oxidative stress and inflammation
Authors: A Kindermann, L Binder, J Baier, B Gündel, A Simm, R Haase, B Bartling
BMC Pulm Med, 2019-12-16;19(1):245.
Species: Mouse
Sample Types: BALF
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Characterization of the seminal plasma proteome in men with prostatitis by mass spectrometry.
Authors: Kagedan D, Lecker I, Batruch I
Clin Proteomics, 2012-02-06;9(1):2.
Species: Rabbit
Sample Types: Seminal Plasma
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Spermatogenic and sperm quality differences in an experimental model of metabolic syndrome and hypogonadal hypogonadism.
Authors: Mallidis C, Czerwiec A, Filippi S, O'Neill J, Maggi M, McClure N
Reproduction, 2011-04-04;142(1):63-71.
Species: Rabbit
Sample Types: Seminal Plasma
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Priming with endotoxin increases acute lung injury in mice by enhancing the severity of lung endothelial injury.
Authors: Lee JS, Su X, Rackley C, Matthay MA, Gupta N
Anat Rec (Hoboken), 2010-11-16;294(1):165-72.
Species: Mouse
Sample Types: BALF
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Ligands of the receptor for advanced glycation end products, including high-mobility group box 1, limit bacterial dissemination during Escherichia coli peritonitis.
Authors: van Zoelen MA, Achouiti A, Schmidt AM, Yang H, Florquin S, Tracey KJ, van der Poll T
Crit. Care Med., 2010-06-01;38(6):1414-22.
Species: Mouse
Sample Types: Peritoneal Fluid
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Oxidative stress-associated rise of hepatic protein glycation increases inflammatory liver injury in uncoupling protein-2 deficient mice.
Authors: Kuhla A, Hettwer C, Menger MD
Lab. Invest., 2010-04-05;90(8):1189-98.
Species: Mouse
Sample Types: Tissue Homogenates
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Disparate effects on renal and oxidative parameters following RAGE deletion, AGE accumulation inhibition, or dietary AGE control in experimental diabetic nephropathy.
Authors: Tan AL, Sourris KC, Harcourt BE, Thallas-Bonke V, Penfold S, Andrikopoulos S, Thomas MC, O'Brien RC, Bierhaus A, Cooper ME, Forbes JM, Coughlan MT
Am. J. Physiol. Renal Physiol., 2009-12-16;298(3):F763-70.
Species: Mouse
Sample Types: Tissue Homogenates
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Ethyl pyruvate decreases HMGB1 release and ameliorates murine colitis.
Authors: Dave SH, Tilstra JS, Matsuoka K, Li F, DeMarco RA, Beer-Stolz D, Sepulveda AR, Fink MP, Lotze MT, Plevy SE
J. Leukoc. Biol., 2009-05-19;86(3):633-43.
Species: Mouse
Sample Types: Feces
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