Human Fibronectin 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
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: Fibronectin
Fibronectin is an extracellular matrix (ECM) component and is one of the primary cell adhesion molecules. The occurrence of different isoforms is due to alternative mRNA splicing of the ED-A, ED-B and III-CS regions, and subsequent post-translational modification. Although non-reactive with adhesion receptors in its soluble state, fibronectin is highly adhesive when immobilized on a surface.
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 Fibronectin 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 - 6
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TMAO enhances TNF-? mediated fibrosis and release of inflammatory mediators from renal fibroblasts
Authors: Stefania, K;Ashok, KK;Geena, PV;Katarina, P;Isak, D;
Scientific reports
Species: Human
Sample Types: Cell Culture Supernates
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Chronic Glaucoma Induced in Rats by a Single Injection of Fibronectin-Loaded PLGA Microspheres: IOP-Dependent and IOP-Independent Neurodegeneration
Authors: Munuera, I;Aragon-Navas, A;Villacampa, P;Gonzalez-Cela, MA;Subías, M;Pablo, LE;Garcia-Feijoo, J;Herrero-Vanrell, R;Garcia-Martin, E;Bravo-Osuna, I;Rodrigo, MJ;
International journal of molecular sciences
Species: Human
Sample Types: Cell Culture Supernates
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CTHRC1 Induces Pancreatic Stellate Cells (PSCs) into Myofibroblast-like Cancer-Associated Fibroblasts (myCAFs)
Authors: Kang, MK;Jiang, F;Kim, YJ;Ryu, K;Masamune, A;Hamada, S;Park, YY;Koh, SS;
Cancers
Species: Human
Sample Types: Cell Culture Supernartes
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Hypoxic Incubation Conditions for Optimized Manufacture of Tenocyte-Based Active Pharmaceutical Ingredients of Homologous Standardized Transplant Products in Tendon Regenerative Medicine
Authors: A Jeannerat, C Peneveyre, F Armand, D Chiappe, R Hamelin, C Scaletta, N Hirt-Burri, A de Buys Ro, W Raffoul, LA Applegate, A Laurent
Cells, 2021-10-25;10(11):.
Species: Human
Sample Types: Cell Lysates
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Platelet-Released Growth Factors and Platelet-Rich Fibrin Induce Expression of Factors Involved in Extracellular Matrix Organization in Human Keratinocytes 22.39
Authors: A Bayer, B Wijaya, L Möbus, F Rademacher, M Rodewald, M Tohidnezha, T Pufe, D Drücke, R Gläser, J Harder
Int J Mol Sci, 2020-06-20;21(12):.
Species: Human
Sample Types: Cell Culture Supernates
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Whole Exome Sequencing of HIV-1 long-term non-progressors identifies rare variants in genes encoding innate immune sensors and signaling molecules
Authors: SK Nissen, M Christians, M Helleberg, K Kjær, SE Jørgensen, J Gerstoft, TL Katzenstei, T Benfield, G Kronborg, CS Larsen, A Laursen, G Pedersen, MR Jakobsen, M Tolstrup, TH Mogensen
Sci Rep, 2018-10-15;8(1):15253.
Species: Human
Sample Types: Plasma
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using supernatants from primary lung fibroblasts
Measurement of fibronectin in cell culture supernatants in ARPE-19 cells with or without stimulation by StemXVivo for 1-5 days
