Human Total EGFR DuoSet IC ELISA

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Product Details
Citations (9)
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Human Total EGFR DuoSet IC ELISA Summary

Assay Type
Solid Phase Sandwich ELISA
96-well strip plate
Assay Length
4 hours 40 mins (after plate preparation)
Sample Volume Required
Cell lysates (100 µL)
Assay Range
31.2 - 2,000 pg/mL
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 total EGFR in cell lysates. An immobilized capture antibody specific for EGFR binds both phosphorylated and unphosphorylated EGFR. After washing away unbound material, a biotinylated detection antibody is used to detect both phosphorylated and unphosphorylated protein, utilizing a standard Streptavidin-HRP format.

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
  • 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
  • Streptavidin-HRP

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: From Costar EIA Plate (Costar Catalog # 2592) or 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

Standard Curve

Figure 1. The Human Total-EGF R DuoSet IC ELISA is more sensitive than immunoprecipitation (IP)-Western blot analysis Lysates prepared from the human epidermoid carcinoma cell line, A431, were diluted in series and analyzed by (A) IP-Western blot and (B) R&D Systems’ Human Total-EGF R DuoSet IC ELISA (Catalog # DYC1854). IPs were performed using anti-EGF R polyclonal antibody (Catalog # AF231) Immunoblots were incubated with a biotinylated anti-EGF R polyclonal antibody (Catalog # BAF231) to detect total EGF R. Blots were incubated with Streptavidin-HRP (Catalog # DY998) followed by chemiluminescent detection. Human EGF R can be detected by the Human Total-EGF R DuoSet IC ELISA by using approximately 200 to 500 times less lysate than is needed for a conventional IP-Western blot.

Figure 2. The Human Total-EGF R DuoSet IC ELISA measures the relative level of EGF R Lysates were prepared from A431 cells, human immortalized fibroblast cells (CCD1070SK), and human breast adenocarcinoma cells (SkBr3). ELISA analysis was done using 125 ng (A431) and 2000 ng (CCD1070SK and SkBr3) of lysate. IP-Western blot analysis (inset) was done using 5 μg (A431) and 100 μg (CCD1070SK and SkBr3) of lysate. The IP-Western blot was performed as described in Figure 1. The smaller band visible in IP-Western blot is an immature EGF R glycosylation intermediate.

Product Datasheets

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Preparation and Storage

The product is shipped at ambient temperature. 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 Total 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.

9 Citations: Showing 1 - 9
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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. Synthesis and biochemical characterization of EGF receptor in a water-soluble membrane model system
    Authors: TM Scharadin, W He, Y Yiannakou, AA Tomilov, M Saldana, GA Cortopassi, KL Carraway, MA Coleman, PT Henderson
    PLoS ONE, 2017;12(6):e0177761.
    Species: Human
    Sample Types: Cell Culture Supernates
  3. 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
  4. Protein shedding in urothelial bladder cancer: prognostic implications of soluble urinary EGFR and EpCAM.
    Authors: Bryan R, Regan H, Pirrie S, Devall A, Cheng K, Zeegers M, James N, Knowles M, Ward D
    Br J Cancer, 2015;112(6):1052-8.
    Species: Human
    Sample Types: Urine
  5. Epidermal growth factor receptor reactivation induced by E-prostanoid-3 receptor- and tumor necrosis factor-alpha-converting enzyme-dependent feedback exaggerates interleukin-8 production in airway cancer (NCI-H292) cells.
    Authors: Kim S, Lewis C, Nadel JA
    Exp. Cell Res., 2011;317(18):2650-60.
    Species: Human
    Sample Types: Cell Lysates
  6. Pertuzumab in combination with trastuzumab shows significantly enhanced antitumor activity in HER2-positive human gastric cancer xenograft models.
    Authors: Yamashita-Kashima Y, Iijima S, Yorozu K, Furugaki K, Kurasawa M, Ohta M, Fujimoto-Ouchi K
    Clin. Cancer Res., 2011;17(15):5060-70.
    Species: Human
    Sample Types: Tissue Homogenates
  7. 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
  8. Identification of biomarkers in human head and neck tumor cell lines that predict for in vitro sensitivity to gefitinib.
    Authors: Hickinson DM, Marshall GB, Beran GJ
    Clin Transl Sci, 2009;2(3):183-92.
    Species: Human
    Sample Types: Cell Lysates
  9. 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


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Human Total EGFR DuoSet IC ELISA
By Balaji Mahender on 01/18/2018
Sample Tested: Cell culture supernatant