Mouse HB-EGF 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: HB-EGF
HB-EGF (Heparin-Binding EGF-like growth factor), also known as DTR, is produced by bronchial epithelium, visceral and vascular smooth muscle, CD4+ T cells, cardiac muscle, glomerular podocytes, keratinocytes, and IL-10-secreting regulatory macrophages. It activates both the EGF R/ErbB1 and ErbB4 receptors and has been linked to many cellular processes including proliferation, apoptosis, cell migration/invasion, differentiation, morphogenesis, and development. HB-EGF is also involved in several aspects of cancer development and progression.
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 HB-EGF 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 - 2
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Semaphorin 4B is an ADAM17-cleaved adipokine that inhibits adipocyte differentiation and thermogenesis
Authors: A Amin, M Badenes, J Tüshaus, É de Carvalh, E Burbridge, P Faísca, K Trávní?kov, A Barros, S Carobbio, PM Domingos, A Vidal-Puig, LF Moita, S Maguire, K St?íšovský, FJ Ortega, JM Fernández-, SF Lichtentha, C Adrain
Molecular Metabolism, 2023-04-28;0(0):101731.
Species: Mouse
Sample Types: Cell Culture Supernates
Applications: ELISA -
Deoxycholic acid activates epidermal growth factor receptor and promotes intestinal carcinogenesis by ADAM17-dependent ligand release
Authors: W Dong, L Liu, Y Dou, M Xu, T Liu, S Wang, Y Zhang, B Deng, B Wang, H Cao
J. Cell. Mol. Med., 2018-06-29;0(0):.
Species: Mouse
Sample Types: Cell Culture Supernates
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Average Rating: 2.5 (Based on 2 Reviews)
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Bottom half of standard curve completely unusable, didn't develop past 4th point on standard curve. This happened across several plates and on three separate occasions. Top OD reached after ~20 mins of incubation with TMB was only about 0.5 (normally 1-2.5).
HB-EGF ELISA was done in parallel to mouse AREG, BTC and EREG and those all worked fine. All reagents were made up in parallel as well.
protocol was fairly easy to follow (you have to keep going back and forth between the handout and the CoA sheet provided in order to make the reagents properly). Unfortunately, there was not a positive control, so I had to use recombinant protein to spike the assay to make sure it worked properly.
