Overview

  • Product name
    Phospho-EGFR (Y1173) + Total In-Cell ELISA Kit
    See all EGFR kits
  • Detection method
    Colorimetric
  • Sample type
    Adherent cells, Suspension cells
  • Assay type
    Cell-based (quantitative)
  • Assay duration
    Multiple steps standard assay
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Product overview

    Phospho-EGFR (Y1173) + Total In-Cell ELISA Kit (ab207463) provides a simple, efficient, cell-based method to monitor proteins activated by phosphorylation. This kit is designed specifically to quantify activated (phosphorylated) EGFR and/or total EGFR. Cells are cultured in 96-well plates and stimulated to induce the pathway of interest. Following stimulation, the cells are rapidly fixed to preserve activation-specific protein modifications. Each well is then incubated with a primary antibody that recognizes either phosphorylated EGFR or total EGFR. Subsequent incubation with secondary HRP-conjugated antibody and developing solution provides an easily quantified colorimetric readout. The relative number of cells in each well is then determined using the provided Crystal Violet solution. The 96-well plate format is suitable for high-throughput screening applications.


    The Phospho-EGFR (Y1173) + Total In-Cell ELISA Kit (ab207463) contains two 96-well plates and two primary antibodies. The phospho-EGFR (Tyr1173) antibody was raised in rabbit against a synthetic phospho-peptide corresponding to residues surrounding phosphorylated Tyr1173 of human EGF receptor. The phospho-EGFR (Tyr1173) antibody recognizes EGFR only when phosphorylated at Tyr1173. The total-EGFR antibody recognizes EGFR protein regardless of its phosphorylation site. This kit can be used to study phosphorylated EGFR relative to cell number or be used to determine EGFR phosphorylation relative to the total EGFR protein found in the cells. Once the phospho-EGFR and total- EGFR signals have been normalized for cell number, a comparison of the ratio of phosphorylated EGFR to total EGFR for each of the cell growth conditions can be made. The provided total-EGFR antibody can be used as a positive control to demonstrate that the cells contain EGFR, the kit reagents are functional and that the protocol is performed correctly. Also, because fixed cells are stable for several weeks, you can prepare many plates simultaneously and then perform the assay when desired.

  • Notes

    Epidermal Growth Factor (EGF) Receptor (EGFR, HER-1, c-ErbB-1) is a member of the Epidermal Growth Factor Receptor family of Receptor Tyrosine Kinases. These cell surface receptors play an important role in the flow of information from the outside of a cell to the inside. Upon binding of EGF to the extracellular domain, the receptor undergoes dimerization and becomes phosphorylated on several tyrosine residues within the cytoplasmic domain. This results in EGFR activation and increased tyrosine kinase activity toward a variety of intracellular substrates. Autophosphorylation of tyrosine 845, 992 and 1173 are critical to EGFR signaling. Phosphorylation at tyrosine 992 creates a direct binding site for phospholipase C-g (PLC-g). PLC-g binding to EGFR (Y992) results in the activation of protein kinase C and the subsequent activation of downstream signaling cascades. Phosphorylation at 1173 creates a major binding site for the protein tyrosine phosphatase SHP-1, which can dephosphorylate the EGFR and thereby block EGFR-induced activation of the ERK1/2 signaling pathway. Tyrosine 845 phosphorylation is mediated by integrin engagement and Src, and regulates receptor function and tumor progression. Aberrant regulation of EGFR signaling has been associated with many types of cancer including mammary, ovarian, non-small cell lung, glioblastoma, prostate, pancreas and head and neck. Thus, methods to rapidly quantify receptor activation are in high demand.

    The Epidermal Growth Factor Receptor (EGFR, HER-1, c-ErbB-1) is a 170 kDa member of the Epidermal Growth Factor (EGF) Receptor family of Receptor Tyrosine Kinases (RTKs) which includes ErbB-2 (Her2/neu), ErbB-3 (HER3) and ErbB-4 (HER4). These cell-surface receptors are glycoproteins comprised of an extracellular ligand-binding domain, a single transmembrane domain and an intracellular domain with ligand-activated tyrosine kinase activity. These proteins modulate critical cellular processes such as mitogenesis, cell death, angiogenesis and cell differentiation.

    Proteins of the EGFR family of RTKs are activated by ligand binding. This results in receptor dimerization, autophosphorylation, activation of downstream signaling molecules and ultimately, lysosomal degradation of the receptor. Natural ligands that activate EGFR are EGF and TGF-alpha.

    EGFR activity is modulated to a large extent through phosphorylation of several phosphorylation and autophosphorylation sites. Autophosphorylation at Tyr992 creates a direct binding site for the SH2 domain of phospholipase C-g (PLC-g). PLC-g binding to EGFR Y992 results in the activation of protein kinase C (PKC) and subsequent activation of the MAP kinase/ERK signaling cascades. These cascades in turn regulate a variety of proteins, including cytosolic proteins involved in protein translation and nuclear proteins such as the transcription factors Elk-1, c-Jun, STAT1 and 3, Ets-1, c-Myc, ER, CREB and PPARg. Phosphorylation at Tyr1173 permits binding of the N-terminal SH2 domain for the protein tyrosine phosphatase SHP-1. SHP-1 has the capacity to activate the cytoplasmic tyrosine kinase c-Src by dephosphorylation of the inhibitory phosphotyrosine at the C-terminus, an event that may be related to SHP-1-dependent activation of the MAP kinase cascade. Tyrosine 845, a major autophosphorylation site in EGFR, is mediated by integrin engagement and Src, and regulates receptor function and tumor progression. Phosphorylation of Tyr845 in the kinase domain may stabilize the activation loop, maintain the enzyme in an active state and provide a binding surface for substrate proteins. c-Src is involved in phosphorylation of Tyr845.

    Aberrant control of EGFR is implicated in development, progression and severity of several human cancers. For example, EGFR overexpression correlates with poor prognosis in breast, ovarian and head/neck cancers. Several mutant forms of the coding gene have also been found. DEGFR, a deletion mutation lacking exons 2-7, is most common and is associated with glioblastoma.

  • Tested applications
    Suitable for: In-Cell ELISAmore details
  • Platform
    Microplate

Properties

  • Storage instructions
    Please refer to protocols.
  • Components 1 x 96 tests 5 x 96 tests
    1% SDS Solution 1 x 22ml 5 x 22ml
    10% Triton X-100 1 x 10ml 5 x 10ml
    10X PBS 1 x 120ml 5 x 120ml
    1X Antibody Blocking Buffer 1 x 22ml 5 x 22ml
    1X Antibody Dilution Buffer 1 x 30ml 5 x 30ml
    96-well tissue culture plate 2 units 10 units
    Anti-rabbit HRP-conjugated IgG 2 x 11µl 10 x 11µl
    Crystal Violet Solution 1 x 22ml 5 x 22ml
    Developing Solution 2 x 11ml 10 x 11ml
    Phospho-EGFR (Y1173) antibody 2 x 11µl 10 x 11µl
    Plate sealer 2 units 10 units
    Stop Solution 2 x 11ml 10 x 11ml
    Total-EGFR antibody 2 x 11µl 10 x 11µl
  • Research areas
  • Function
    Receptor tyrosine kinase binding ligands of the EGF family and activating several signaling cascades to convert extracellular cues into appropriate cellular responses. Known ligands include EGF, TGFA/TGF-alpha, amphiregulin, epigen/EPGN, BTC/betacellulin, epiregulin/EREG and HBEGF/heparin-binding EGF. Ligand binding triggers receptor homo- and/or heterodimerization and autophosphorylation on key cytoplasmic residues. The phosphorylated receptor recruits adapter proteins like GRB2 which in turn activates complex downstream signaling cascades. Activates at least 4 major downstream signaling cascades including the RAS-RAF-MEK-ERK, PI3 kinase-AKT, PLCgamma-PKC and STATs modules. May also activate the NF-kappa-B signaling cascade. Also directly phosphorylates other proteins like RGS16, activating its GTPase activity and probably coupling the EGF receptor signaling to the G protein-coupled receptor signaling. Also phosphorylates MUC1 and increases its interaction with SRC and CTNNB1/beta-catenin.
    Isoform 2 may act as an antagonist of EGF action.
  • Tissue specificity
    Ubiquitously expressed. Isoform 2 is also expressed in ovarian cancers.
  • Involvement in disease
    Lung cancer
    Inflammatory skin and bowel disease, neonatal, 2
  • Sequence similarities
    Belongs to the protein kinase superfamily. Tyr protein kinase family. EGF receptor subfamily.
    Contains 1 protein kinase domain.
  • Post-translational
    modifications
    Phosphorylation at Ser-695 is partial and occurs only if Thr-693 is phosphorylated. Phosphorylation at Thr-678 and Thr-693 by PRKD1 inhibits EGF-induced MAPK8/JNK1 activation. Dephosphorylation by PTPRJ prevents endocytosis and stabilizes the receptor at the plasma membrane. Autophosphorylation at Tyr-1197 is stimulated by methylation at Arg-1199 and enhances interaction with PTPN6. Autophosphorylation at Tyr-1092 and/or Tyr-1110 recruits STAT3. Dephosphorylated by PTPN1 and PTPN2.
    Monoubiquitinated and polyubiquitinated upon EGF stimulation; which does not affect tyrosine kinase activity or signaling capacity but may play a role in lysosomal targeting. Polyubiquitin linkage is mainly through 'Lys-63', but linkage through 'Lys-48', 'Lys-11' and 'Lys-29' also occurs. Deubiquitination by OTUD7B prevents degradation. Ubiquitinated by RNF115 and RNF126.
    Methylated. Methylation at Arg-1199 by PRMT5 stimulates phosphorylation at Tyr-1197.
  • Cellular localization
    Secreted and Cell membrane. Endoplasmic reticulum membrane. Golgi apparatus membrane. Nucleus membrane. Endosome. Endosome membrane. Nucleus. In response to EGF, translocated from the cell membrane to the nucleus via Golgi and ER. Endocytosed upon activation by ligand. Colocalized with GPER1 in the nucleus of estrogen agonist-induced cancer-associated fibroblasts (CAF).
  • Information by UniProt
  • Alternative names
    • Avian erythroblastic leukemia viral (v erb b) oncogene homolog
    • Cell growth inhibiting protein 40
    • Cell proliferation inducing protein 61
    • EGF R
    • EGFR
    • EGFR_HUMAN
    • Epidermal growth factor receptor
    • Epidermal growth factor receptor (avian erythroblastic leukemia viral (v erb b) oncogene homolog)
    • Epidermal growth factor receptor (erythroblastic leukemia viral (v erb b) oncogene homolog avian)
    • erb-b2 receptor tyrosine kinase 1
    • ERBB
    • ERBB1
    • Errp
    • HER1
    • mENA
    • NISBD2
    • Oncogen ERBB
    • PIG61
    • Proto-oncogene c-ErbB-1
    • Receptor tyrosine protein kinase ErbB 1
    • Receptor tyrosine-protein kinase ErbB-1
    • SA7
    • Species antigen 7
    • Urogastrone
    • v-erb-b Avian erythroblastic leukemia viral oncogen homolog
    • wa2
    • Wa5
    see all
  • Database links

Applications

Our Abpromise guarantee covers the use of ab207463 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
In-Cell ELISA Use at an assay dependent concentration.

Protocols

References

ab207463 has not yet been referenced specifically in any publications.

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