Human Phospho-EGFR DuoSet IC ELISA
R&D Systems, part of Bio-Techne | Catalog # DYC1095B-2
Key Product Details
Assay Type
Solid Phase Sandwich ELISA
Sample Type
Cell Lysates
Reactivity
Human
Human Phospho-EGFR 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-EGFR DuoSet IC ELISA
This DuoSet IC ELISA contains the basic components required for the development of sandwich ELISAs to measure tyrosine-phosphorylated human EGFR in cell lysates. An immobilized capture antibody specific for EGFR binds both phosphorylated and unphosphorylated EGFR . After washing away unbound material, an HRP-conjugated 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-EGFR DuoSet IC ELISA
Figure 1: The Human Phospho-EGF R DuoSet IC ELISA is more sensitive than immunoprecipitation (IP)-Western blot analysis
The human epidermoid carcinoma cell line, A431, was treated with 25 ng/mL recombinant human EGF (R&D Systems Catalog #236-EG) for 5 minutes to induce tyrosine phosphorylation of EGF R. Lysates were serially diluted and analyzed by (A) IP-Western blot and (B) this DuoSet IC ELISA. IPs were done using an anti-EGF R monoclonal antibody and anti-mouse IgG agarose. Immunoblots were incubated with an HRP-conjugated anti-phospho-tyrosine monoclonal antibody (R&D Systems, Catalog #HAM1676) to detect phosphor-EGF R. Bands were visualized by chemiluminescent detection. Human Phospho-EGF R can be detected in this DuoSet IC ELISA by using approximately 2-4 times less lysate than is needed for a conventional IP-Western blot.
Figure 2: The Human Phospho-EGF R DuoSet IC ELISA detects ligand-induced EGF R tyrosine phosphorylation
A431 cells were untreated or treated with 25 ng/mL recombinant human EGF for 5 minutes. ELISA and IP-Western blot (inset) analyses were done using 25 and 50 μg of lysate, respectively. IP-Western blots for phospho-EGF R (p-EGF R) were done as described in Figure 1. Blots were stripped and total EGF R was detected using a biotinylated anti-EGF R polyclonal antibody (R&D Systems, Catalog #AF231).
Figure 3. The quantification of Human Phospho-EGF R DuoSet IC ELISA is demonstrated by using cells pretreated with the inhibitor PD168393
A431 cells were incubated with no additions or treated with 25 ng/mL recombinant human EGF for 5 minutes, after either being treated with or without 1 nM, 10 nM, or 100 nM PD168393. ELISA and IP-Western blot (inset) analyses were done using 25 and 50 μg of lysate, respectively. IP-Western blots for phospho-EGF R (p-EGF R) were done as described in Figure 1. Total EGF R blots were done as described in Figure 2. The DuoSet IC ELISA results correlate well with the relative amount of phosphorylated EGF R detected by IP-Western blot.Kit Contents for Human Phospho-EGFR 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: EGFR
EGFR (epidermal growth factor receptor), also known as HER-1, ErbB1, or ErbB, is a transmembrane receptor tyrosine kinase that binds a subset of the EGF family ligands including EGF, Amphiregulin, TGF-alpha, Betacellulin, Epiregulin, HB-EGF, and Epigen. Ligand binding induces EGFR homodimerization as well as heterdimerization with ErbB2, resulting in kinase activation, tyrosine phosphorylation and cell signaling. EGFR can also be recruited to form heterodimers with the ligand-activated ErbB3 or ErbB4. EGFR signaling regulates multiple biological functions including cell proliferation, differentiation, motility, and apoptosis. EGFR is overexpressed in a wide variety of tumors and is the target of several anti-cancer drugs. Soluble receptors consisting of the extracellular ligand binding domain are generated by alternate splicing in human and mouse.
Long Name
Epidermal Growth Factor Receptor
Alternate Names
EGF R, ErbB, ErbB1, HER-1
Gene Symbol
EGFR
Additional EGFR Products
Product Documents for Human Phospho-EGFR DuoSet IC ELISA
Product Specific Notices for Human Phospho-EGFR DuoSet IC ELISA
For research use only
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