Mouse Wnt-3a 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
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
Block Buffer: (Catalog # DY995), or 1% BSA in PBS, pH 7.2-7.4, 0.2 µm filtered
Reagent Diluent: 0.1% BSA, 0.05% Tween 20 in Tris-buffered Saline (20 mM Trizma base, 150 mM NaCI) 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)
*For the Reagent Diluent and Blocking Buffer recommended for a specific DuoSet ELISA Development Kit, please see the product.
Product Datasheets
Preparation and Storage
Background: Wnt-3a
Wnt-3a is a secreted protein that is necessary for proper embryonic development of the hippocampus, anterior-posterior patterning, somite development, and tailbud formation. It also promotes self-renewal of hematopoietic stem cells, neural stem cells, and embryonic stem cells. The post-translational glycosylation and acylation of Wnts are essential for their efficient secretion and biological activity, respectively. Wnts bind to the cell surface Frizzled family receptors in conjunction with low-density lipoprotein receptor-related protein family receptors (LRP5 or 6) resulting in the stabilization of intracellular beta-catenin levels. As intracellular beta-catenin levels rise, beta-catenin binds to TCF/LEF transcription factors leading to expression of Wnt target genes.
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 of Block Buffer 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 Wnt-3a 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 - 3
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Chronic expression of p16INK4a in the epidermis induces Wnt-mediated hyperplasia and promotes tumor initiation
Authors: N Azazmeh, B Assouline, E Winter, S Ruppo, Y Nevo, A Maly, K Meir, AK Witkiewicz, J Cohen, SV Rizou, E Pikarsky, C Luxenburg, VG Gorgoulis, I Ben-Porath
Nat Commun, 2020-06-01;11(1):2711.
Species: Mouse
Sample Types: Cell Culture Supernates
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Attenuation of Wnt/beta-catenin signaling in patients with Stevens-Johnson syndrome and toxic epidermal necrolysis
Authors: CB Chen, WC Chang, MY Wu, TY Kao, YW Wang, CW Wang, CJ Chen, WH Chung, SC Su
Int. J. Biol. Sci., 2020-01-01;16(2):353-364.
Species: Human
Sample Types: Serum
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Fenretinide, Tocilizumab & Reparixin Provide Multifaceted Disruption of Oral Squamous Cell Carcinoma Stem Cell Properties: Implications for Tertiary chemoprevention
Authors: SR Mallery, D Wang, B Santiago, P Pei, C Bissonnett, JA Jayawarden, SP Schwendema, R Spinney, J Lang
Mol. Cancer Ther., 2019-09-12;0(0):.
Species: Human
Sample Types: Cell Culture Supernates
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