Human Phospho-ErbB3/Her3 DuoSet IC ELISA
R&D Systems, part of Bio-Techne | Catalog # DYC1769-2
Key Product Details
Assay Type
Solid Phase Sandwich ELISA
Sample Type
Cell Lysates
Reactivity
Human
Human Phospho-ErbB3/Her3 DuoSet IC ELISA 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
Product Summary for Human Phospho-ErbB3/Her3 DuoSet IC ELISA
This DuoSet IC ELISA contains the basic components required for the development of sandwich ELISAs to measure tyrosine-phosphorylated human ErbB3 / Her3 in cell lysates. An immobilized capture antibody specific for ErbB3 / Her3 binds both phosphorylated and unphosphorylated ErbB3 / Her3 . After washing away unbound material, a biotinylated detection antibody is used to detect only phosphorylated receptor, utilizing a standard HRP format.
Product Specifications
Assay Format
96-well strip plate (sold separately)
Sample Volume Required
Cell lysates (100 µL)
Detection Method
Colorimetric ELISA - 450nm (TMB)
Conjugate
Biotin
Specificity
Label
HRP
Scientific Data Images for Human Phospho-ErbB3/Her3 DuoSet IC ELISA
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.
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).
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.Kit Contents for Human Phospho-ErbB3/Her3 DuoSet IC ELISA
- 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
Preparation and Storage
Shipping
The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.
Background: ErbB3/Her3
Long Name
Receptor Tyrosine Protein Kinase ErbB3
Alternate Names
HER3
Gene Symbol
ERBB3
Additional ErbB3/Her3 Products
Product Documents for Human Phospho-ErbB3/Her3 DuoSet IC ELISA
Product Specific Notices for Human Phospho-ErbB3/Her3 DuoSet IC ELISA
For research use only
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