Human Phospho-EGFR DuoSet IC ELISA

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Ancillary Products Available
Figure 1: The Human Phospho-EGF R DuoSet IC ELISA is more sensitive than immunoprecipitation (IP)-Western blot analysis
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Product Details
Citations (14)
Supplemental Products

Human Phospho-EGFR DuoSet IC ELISA Summary

Assay Type
Solid Phase Sandwich ELISA
96-well strip plate
Sample Volume Required
Cell lysates (100 µL)
Sufficient Materials
Kits available for two, five, or fifteen 96-well plates*
Please see the product datasheet

* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.

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 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

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.

Figure 4. The specificity of Human Phospho-EGF R DuoSet IC ELISA is confirmed by receptor competition A431 cells were treated with 25 ng/mL recombinant human EGF for 5 minutes. The indicated amounts of recombinant extracellular domains of human EGF R (R&D Systems, Catalog #1095-ER), human ErbB2/Fc Chimera (R&D Systems, Catalog #1129-ER), human ErbB3/Fc Chimera (R&D Systems, Catalog #348-RB), or human ErbB4/Fc Chimera (R&D Systems, Catalog #1131-ER) were added to 25 μg of lysate and analyzed using this DuoSet IC ELISA. Competition was observed only with recombinant human EGF R.

Product Datasheets

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Preparation and Storage

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

The EGF R subfamily of receptor tyrosine kinases comprises four members: EGF R (also known as HER-1, ErbB1, or ErbB), ErbB2 (Neu, HER-2), ErbB3 (HER-3), and ErbB4 (HER-4). All family members are type I transmembrane glycoproteins with an extracellular ligand binding domain containing two cysteine-rich domains separated by a spacer region and a cytoplasmic domain containing a membrane-proximal tyrosine kinase domain followed by multiple tyrosine autophosphorylation sites. The human EGF R cDNA encodes a 1210 amino acid (aa) precursor with a 24 aa signal peptide, a 621 aa extracellular domain (ECD), a 23 aa transmembrane segment, and a 542 aa cytoplasmic domain. Soluble receptors consisting of the extracellular ligand binding domain are generated by alternate splicing in human and mouse. Within the ECD, human EGF R shares 88% aa sequence identity with mouse and rat EGF R. It shares 43% - 44% aa sequence identity with the ECD of human ErbB2, ErbB3, and ErbB4. EGF R binds a subset of the EGF family ligands, including EGF, amphiregulin, TGF-alpha, betacellulin, epiregulin, HB-EGF, and epigen. Ligand binding induces EGF R homodimerization as well as heterodimerization with ErbB2, resulting in kinase activation, heterodimerization tyrosine phosphorylation and cell signaling. EGF R can also be recruited to form heterodimers with the ligand-activated ErbB3 or ErbB4. EGF R signaling regulates multiple biological functions including cell proliferation, differentiation, motility, and apoptosis. EGF R is overexpressed in a wide variety of tumors and is the target of several anti-cancer drugs.

Long Name:
Epidermal Growth Factor Receptor
Entrez Gene IDs:
1956 (Human); 13649 (Mouse); 24329 (Rat); 102138724 (Cynomolgus Monkey)
Alternate Names:
avian erythroblastic leukemia viral (v-erb-b) oncogene homolog; cell growth inhibiting protein 40; cell proliferation-inducing protein 61; EC 2.7.10; EC; EGF R; EGFR; epidermal growth factor receptor (avian erythroblastic leukemia viral (v-erb-b)oncogene homolog); epidermal growth factor receptor; ErbB; ErbB1; ERBB1PIG61; HER1; HER-1; mENA; Proto-oncogene c-ErbB-1; Receptor tyrosine-protein kinase erbB-1

Citations for Human Phospho-EGFR 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.

14 Citations: Showing 1 - 10
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  1. Anti-tumor activity of osimertinib, an irreversible mutant-selective EGFR tyrosine kinase inhibitor, in NSCLC harboring EGFR Exon 20 Insertions
    Authors: N Floc'h, MJ Martin, JW Riess, JP Orme, AD Staniszews, L Menard, ME Cuomo, DJ O'Neill, RA Ward, MRV Finlay, D McKerreche, M Cheng, DP Vang, RA Tsai, JG Keck, DR Gandara, PC Mack, DA Cross
    Mol. Cancer Ther., 2018;0(0):.
    Species: Human
    Sample Types: Tissue Homogenates
  2. Andrographolide enhanced 5-fluorouracil-induced antitumor effect in colorectal cancer via inhibition of c-MET pathway
    Authors: M Su, B Qin, F Liu, Y Chen, R Zhang
    Drug Des Devel Ther, 2017;11(0):3333-3341.
    Species: Human
    Sample Types: Cell Culture Supernates
  3. Stand-Sit Microchip for High-Throughput, Multiplexed Analysis of Single Cancer Cells
    Sci Rep, 2016;6(0):32505.
    Applications: ChIP
  4. Cetuximab sensitivity of head and neck squamous cell carcinoma xenografts is associated with treatment-induced reduction of EGFR, pEGFR, and pSrc
    Authors: Adam Jedlinski
    J. Oral Pathol. Med, 2016;0(0):.
    Species: Human
    Sample Types: Cell Lysates
  5. The HER2-binding affibody molecule (Z(HER2ratio342))(2) increases radiosensitivity in SKBR-3 cells.
    Authors: Ekerljung L, Lennartsson J, Gedda L
    PLoS ONE, 2012;7(11):e49579.
    Species: Human
    Sample Types: Cell Lysates
  6. Vandetanib inhibits both VEGFR-2 and EGFR signalling at clinically relevant drug levels in preclinical models of human cancer.
    Authors: Brave SR, Odedra R, James NH
    Int. J. Oncol., 2011;39(1):271-8.
    Species: Human
    Sample Types: Cell Lysates
  7. CCL20/CCR6 feedback exaggerates epidermal growth factor receptor-dependent MUC5AC mucin production in human airway epithelial (NCI-H292) cells.
    Authors: Kim S, Lewis C, Nadel JA
    J. Immunol., 2011;186(6):3392-400.
    Species: Human
    Sample Types: Cell Lysates
  8. Lithocholic acid is an Eph-ephrin ligand interfering with Eph-kinase activation.
    Authors: Giorgio C, Hassan Mohamed I, Flammini L, Barocelli E, Incerti M, Lodola A, Tognolini M
    PLoS ONE, 2011;6(3):e18128.
    Species: Human
    Sample Types: Cell Lysates
  9. 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;16(4):1159-69.
    Species: Human
    Sample Types: Cell Lysates
  10. Activated phosphoinositide 3-kinase/AKT signaling confers resistance to trastuzumab but not lapatinib.
    Authors: O'Brien NA, Browne BC, Chow L, Wang Y, Ginther C, Arboleda J, Duffy MJ, Crown J, O'Donovan N, Slamon DJ
    Mol. Cancer Ther., 2010;9(6):1489-502.
    Species: Human
    Sample Types: Cell Lysates
  11. Fibrinogen binding to ICAM-1 promotes EGFR-dependent mucin production in human airway epithelial cells.
    Authors: Kim S, Nadel JA
    Am. J. Physiol. Lung Cell Mol. Physiol., 2009;297(1):L174-83.
    Species: Human
    Sample Types: Cell Lysates
  12. Quantifying the effects of co-expressing EGFR and HER2 on HER activation and trafficking.
    Authors: Shankaran H, Zhang Y, Opresko L, Resat H
    Biochem. Biophys. Res. Commun., 2008;371(2):220-4.
    Species: Human
    Sample Types: Cell Lysates
  13. EGFR signaling is required for TGF-beta 1 mediated COX-2 induction in human bronchial epithelial cells.
    Authors: Liu M, Yang SC, Sharma S, Luo J, Cui X, Peebles KA, Huang M, Sato M, Ramirez RD, Shay JW, Minna JD, Dubinett SM
    Am. J. Respir. Cell Mol. Biol., 2007;37(5):578-88.
    Species: Human
    Sample Types: Cell Lysates
  14. In-frame deletion in the EGF receptor alters kinase inhibition by gefitinib.
    Authors: Sakai K, Yokote H, Murakami-Murofushi K, Tamura T, Saijo N, Nishio K
    Biochem. J., 2006;397(3):537-43.
    Species: Human
    Sample Types: Cell Lysates


  1. Which phosphorylated sites are recognized in this assay?

    • This assay utilizes an anti-phosphorylated tyrosine monoclonal detection antibody, and it recognizes all phosphorylated tyrosines.

View all ELISA FAQs

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