Human Phospho-ErbB3/Her3 DuoSet IC 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 and recommended concentrations save lengthy development time
- Development protocols are provided to guide further assay optimization
- Assay can be customized to your specific needs
- Available in 2, 5, and 15-(96-well) plate pack sizes
- Economical alternative to Western blot
Kit Content
- Capture Antibody
- Conjugated Detection Antibody
- Calibrated Immunoassay Standard or Control
Other Reagents Required
PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2O4, pH 7.2 - 7.4, 0.2 µm filtered
Wash Buffer: (Catalog # WA126), or equivalent
Lysis Buffer*
IC Diluent*
Blocking Buffer*
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
*For the Lysis Buffer, IC Diluent, and Blocking BUffer recommended for a specific DuoSet ELISA Development Kit, please see the product
Scientific Data
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Figure 1. The Human Phospho-ErbB3 DuoSet IC ELISA is more sensitive than immunoprecipitation (IP)-Western blot analysis The human breast cancer cell line, MDA-MB-453, was treated with 100 ng/mL recombinant HRG-beta 1 (Catalog # 396-HB) for five minutes to induce tyrosine phosphorylation of ErbB3. Serial dilutions of lysates were analyzed by R&D Systems’ Phospho-ErbB3 DuoSet IC ELISA (Catalog # DYC1769) and by IP-Western blot (inset). For IP-Westerns, IPs were done using anti-ErbB3 monoclonal antibody and anti-mouse IgG agarose. Immunoblots were incubated with biotinylated anti-phosphotyrosine monoclonal antibody (Cat # BAM1676) to detect phospho-ErbB3. Bands were visualized with Streptavidin-HRP (Cat # DY998) followed by ECL detection. ErbB3 was observed at its predicted size (inset; 185 kDa, arrow) in Western blot analysis.
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Figure 2. The Human Phospho-ErbB3 DuoSet IC ELISA detects ligand-induced ErbB3 tyrosine phosphorylation MDA-MB-453 cells were untreated or treated with 100 ng/mL of recombinant HRG-beta 1 (Catalog # 396-HB) for five minutes. Cell lysates (100 μg) were analyzed by R&D Systems’ Phospho-ErbB3 DuoSet IC ELISA (Catalog # DYC1769) and by IP-Western blot (inset). IP-Western blots for phospho-ErbB3 (p-ErbB3) were done as described in Figure 1. Blots were stripped and total ErbB3 (ErbB3) was detected using biotinylated anti-ErbB3 polyclonal antibody (Catalog # BAF234).
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Figure 3. The specificity of the Human Phospho-ErbB3 DuoSet IC ELISA is confirmed by receptor competition MDA-MB-453 cells were treated with 100 ng/mL of recombinant HRG-beta 1 (Catalog # 396-HB) for five minutes. The indicated amounts of ErbB3 (Catalog # 348-ER), ErbB2 (Catalog # 1129-ER) or EGF R (Catalog # 1095-ER) recombinant extracellular domains were added to 5 μg lysate and analyzed using R&D Systems’ Human Phospho-ErbB3 DuoSet IC ELISA (Catalog # DYC1769). Competition was observed only with recombinant ErbB3.
Product Datasheets
Preparation and Storage
Background: ErbB3/Her3
ErbB3, also called Her3 (human epidermal growth factor receptor 3) in humans, is a type I transmembrane glycoprotein that is a member of the ErbB family of tyrosine kinase receptors. ErbB3 binds neuregulins NRG1 and NRG2, but lacks a working kinase domain; heterodimerization with another ErbB receptor is required for signaling. ErbB3 is important in development of Schwann cells, neural crest cells, heart valves and the sympathetic nervous system. When expressed with ErbB2 in breast, skin, ovary and gastrointestinal tumors, ErbB3 is a potent mitogen.
Citations for Human Phospho-ErbB3/Her3 DuoSet IC 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 - 4
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Evaluating the Therapeutic Efficacy of Mono- and Bivalent Affibody-Based Fusion Proteins Targeting HER3 in a Pancreatic Cancer Xenograft Model
Authors: CD Leitao, SS Rinne, M Altai, O Vorontsova, F Dunås, P Jonasson, V Tolmachev, J Löfblom, S Ståhl, A Orlova
Pharmaceutics, 2020-06-13;12(6):.
Species: Human
Sample Types: Cell Lysates
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The HER2-binding affibody molecule (Z(HER2ratio342))(2) increases radiosensitivity in SKBR-3 cells.
Authors: Ekerljung L, Lennartsson J, Gedda L
PLoS ONE, 2012-11-14;7(11):e49579.
Species: Human
Sample Types: Cell Lysates
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Cellular Effects of HER3-Specific Affibody Molecules.
Authors: Gostring L, Malm M, Hoiden-Guthenberg I, Frejd FY, Stahl S, Lofblom J, Gedda L
PLoS ONE, 2012-06-29;7(6):e40023.
Species: Human
Sample Types: Cell Lysates
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AZD8931, an equipotent, reversible inhibitor of signaling by epidermal growth factor receptor, ERBB2 (HER2), and ERBB3: a unique agent for simultaneous ERBB receptor blockade in cancer.
Authors: Hickinson DM, Klinowska T, Speake G
Clin. Cancer Res., 2010-02-09;16(4):1159-69.
Species: Human
Sample Types: Cell Lysates
FAQs
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Which phosphorylated sites are recognized by this assay?
This assay utilizes an anti-phosphorylated tyrosine monoclonal detection antibody, and it recognizes all phosphorylated tyrosine residues.
Reviews for Human Phospho-ErbB3/Her3 DuoSet IC ELISA
Average Rating: 5 (Based on 2 Reviews)
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