Human sFRP-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.
This DuoSet ELISA Development kit contains the basic components required for the development of sandwich ELISAs to measure natural and recombinant human sFrizzled-related Protein-3/sFRP-3. 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: sFRP-3
Secreted Frizzled related proteins are secreted glycoproteins with homology to the Wnt-binding domain of the Frizzled family of transmembrane receptors. sFRPs contain an N-terminal Frizzled-like cysteine-rich domain (CRD) with 10 conserved cysteines and a C-terminal heparin-binding region. sFRPs function as soluble antagonists of Wnt signaling by binding directly to Wnt proteins and preventing their interactions with Frizzled receptors.
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 Human sFRP-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 - 10
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Secreted Wnt antagonists in scrub typhus
Authors: T Ueland, E Astrup, K Otterdal, T Lekva, J Janardhana, JAJ Prakash, K Thomas, AE Michelsen, P Aukrust, GM Varghese, JK Damås
PloS Neglected Tropical Diseases, 2021-04-29;15(4):e0009185.
Species: Human
Sample Types: Plasma
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Wnt antagonist FRZB is a muscle biomarker of denervation atrophy in amyotrophic lateral sclerosis
Authors: T Kwan, M Kazamel, K Thoenes, Y Si, N Jiang, PH King
Sci Rep, 2020-10-07;10(1):16679.
Species: Human
Sample Types: Cell Lysates
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Dickkopf-related protein 1 and Gremlin 1 show different response than Frizzled-related protein in human synovial fluid following knee injury and in patients with osteoarthritis
Authors: X Huang, JN Post, L Zhong, J Leijten, S Larsson, M Karperien, A Struglics
Osteoarthr. Cartil., 2018-03-08;0(0):.
Species: Human
Sample Types: Synovial Fluid
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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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Nitric Oxide Mediates Crosstalk between Interleukin 1? and WNT Signaling in Primary Human Chondrocytes by Reducing DKK1 and FRZB Expression
Authors: L Zhong, S Schivo, X Huang, J Leijten, M Karperien, JN Post
Int J Mol Sci, 2017-11-22;18(11):.
Species: Human
Sample Types: Cell Culture Supernates
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Wnt5a is associated with right ventricular dysfunction and adverse outcome in dilated cardiomyopathy
Authors: A Abraityte, IG Lunde, ET Askevold, AE Michelsen, G Christense, P Aukrust, A Yndestad, A Fiane, A Andreassen, S Aakhus, CP Dahl, L Gullestad, K Broch, T Ueland
Sci Rep, 2017-06-14;7(1):3490.
Species: Human
Sample Types: Plasma
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Secreted Frizzled Related Protein 3 in Chronic Heart Failure: Analysis from the Controlled Rosuvastatin Multinational Trial in Heart Failure (CORONA).
Authors: Askevold E, Gullestad L, Nymo S, Kjekshus J, Yndestad A, Latini R, Cleland J, McMurray J, Aukrust P, Ueland T
PLoS ONE, 2015-08-19;10(8):e0133970.
Species: Human
Sample Types: Serum
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Metabolic programming of mesenchymal stromal cells by oxygen tension directs chondrogenic cell fate.
Authors: Leijten, Jeroen, Georgi, Nicole, Moreira Teixeira, Liliana, van Blitterswijk, Clemens, Post, Janine N, Karperien, Marcel
Proc Natl Acad Sci U S A, 2014-09-09;111(38):13954-9.
Species: Human
Sample Types: Cell Culture Supernates
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Hypoxia inhibits hypertrophic differentiation and endochondral ossification in explanted tibiae.
Authors: Leijten, Jeroen C, Moreira Teixeira, Liliana, Landman, Ellie B, van Blitterswijk, Clemens, Karperien, Marcel
PLoS ONE, 2012-11-21;7(11):e49896.
Species: Mouse
Sample Types: Cell Culture Supernates
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Methylation and loss of secreted frizzled-related protein 3 enhances melanoma cell migration and invasion.
Authors: Ekstrom EJ, Sherwood V, Andersson T
PLoS ONE, 2011-04-08;6(4):e18674.
Species: Human
Sample Types: Cell Culture Supernates
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Very clear instruction and everything is provided with the kit. Very satisfied.
