Human Dkk-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.
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: Dkk-3
Members of the dickkopf-related protein family (Dkk-1, -2, -3, and -4) are secreted proteins with two cysteine-rich domains separated by a linker region. Dkk-3 and -4 also have one prokineticin domain. Dkk-1, -2, and -4 function as antagonists of canonical Wnt signaling by binding to LRP5/6, preventing interaction of LRP5/6 with Wnt-Frizzled complexes. Dkk-1, -2, and -4 also bind cell surface Kremen-1 or -2 and promote the internalization of LRP5/6. Antagonistic activity of Dkk-3 has not been demonstrated. Dkk proteins have distinct patterns of expression in adult and embryonic tissues and have a wide range of effects on tissue development and morphogenesis. The Dkk family also includes Soggy, which is homologous to Dkk-3 but not to the other family members. Soggy has not been shown to inhibit Wnt signaling, and its role in the pathway is unclear.
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 Human Dkk-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 - 7
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Antibody microarray analysis of amniotic fluid proteomes in women with cervical insufficiency and short cervix, and their association with pregnancy latency length
Authors: S Hong, KH Park, YE Lee, JE Lee, YM Kim, E Joo, I Cho
PLoS ONE, 2022-02-07;17(2):e0263586.
Species: Human
Sample Types: Amniotic Fluid
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The emerging plasma biomarker Dickkopf-3 (DKK3) and its association with renal and cardiovascular disease in the general population
Authors: A Piek, L Smit, N Suthahar, SJL Bakker, RA de Boer, HHW Silljé
Scientific Reports, 2021-04-21;11(1):8642.
Species: Human
Sample Types: Plasma
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Combined Serum DKK3 and Circulating CD133 Cells as Prognostic Biomarkers for Ovarian Cancer Patients
Authors: XC Nie, F He, C Lan, JM Niu, P Xia
OncoTargets and Therapy, 2021-01-14;14(0):427-434.
Species: Human
Sample Types: Serum
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Exploring the Wnt signaling pathway in schizophrenia and bipolar disorder
Authors: EZ Hoseth, F Krull, I Dieset, RH Mørch, S Hope, ES Gardsjord, NE Steen, I Melle, HR Brattbakk, VM Steen, P Aukrust, S Djurovic, OA Andreassen, T Ueland
Transl Psychiatry, 2018-03-06;8(1):55.
Species: Human
Sample Types: Plasma
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Blood-based protein biomarker panel for the detection of colorectal cancer.
Authors: Fung, Kim Y C, Tabor, Bruce, Buckley, Michael, Priebe, Ilka K, Purins, Leanne, Pompeia, Celine, Brierley, Gemma V, Lockett, Trevor, Gibbs, Peter, Tie, Jeanne, McMurrick, Paul, Moore, James, Ruszkiewicz, Andrew, Nice, Edouard, Adams, Timothy, Burgess, Antony, Cosgrove, Leah J
PLoS ONE, 2015-03-20;10(3):e0120425.
Species: Human
Sample Types: Serum
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The clinicopathological significance of REIC expression in colorectal carcinomas.
Authors: Wang W, Zhu W, Xu XY
Histol. Histopathol., 2012-06-01;27(6):735-43.
Species: Human
Sample Types: Cell Culture Supernates
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Integrated proteomic analysis of human cancer cells and plasma from tumor bearing mice for ovarian cancer biomarker discovery.
Authors: Pitteri SJ, JeBailey L, Faca VM, Thorpe JD, Silva MA, Ireton RC, Horton MB, Wang H, Pruitt LC, Zhang Q, Cheng KH, Urban N, Hanash SM, Dinulescu DM
PLoS ONE, 2009-11-19;4(11):e7916.
Species: Human
Sample Types: Plasma
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