Overview

  • Product name
    EGFR (Tyr845) In-Cell ELISA Kit
    See all EGFR kits
  • Detection method
    Colorimetric
  • Sample type
    Adherent cells
  • Assay type
    Cell-based (qualitative)
  • Assay time
    5h 10m
  • Assay duration
    Multiple steps standard assay
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Product overview

    ab126421 is a very rapid, convenient and sensitive assay kit that can monitor the activation or function of important biological pathways in cells. It can be used for measuring the relative amount of EGFR (Tyr845) phosphorylation and screening the effects of various treatments, inhibitors (such as siRNA or chemicals), or activators in cultured Human, Mouse and Rat cell lines. By determining EGFR protein phosphorylation in your experimental model system, you can verify pathway activation in your cell lines without spending excess time and effort in preparing cell lysate and performing an analysis of Western Blot.


    In the EGFR (Tyr845) In-Cell ELISA Kit, cells are seeded into a 96 well tissue culture plate. The cells are fixed after various treatments, inhibitors or activators. After blocking, anti-Phospho-EGFR (Tyr845) or anti-EGFR (primary antibody) is pipetted into the wells and incubated. The wells are washed, and HRP-conjugated secondary antibody is added to the wells. The wells are washed again, a TMB substrate solution is added to the wells and color develops in proportion to the amount of protein. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

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

Properties

  • Storage instructions
    Store at -20°C. Please refer to protocols.
  • Components 1 x 96 tests
    HRP-conjugated Anti-Mouse IgG Concentrate 1 x 10µl
    Anti-Rabbit IgG Concentrate (Item I-1) 1 x 10µl
    Blocking Buffer Concentrate (5X) 1 x 20ml
    Fixing Solution 1 x 30ml
    Uncoated 96-well Microplate 1 unit
    Mouse anti-Phospho-EGFR (Tyr845) Concentrate (Item G) 1 x 6µl
    Quenching Buffer Concentrate (30x) 1 x 2ml
    Rabbit anti-EGFR Concentrate (Item H) 1 x 6µl
    Stop Solution 1 x 14ml
    TMB One-Step Substrate Reagent 1 x 12ml
    Wash Buffer A Concentrate (20X) 1 x 30ml
    Wash Buffer B Concentrate (20X) 1 x 30ml
  • 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 ab126421 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.

Images

  • A431 cells were stimulated by different concentrations of EGF for 10 min at 37°C.

  • Western blot analysis of extracts from 100 ng/ml hEGF treated A431 cells. Phospho-EGFR (Tyr845) and EGFR antibodies were used in both detection assays.
  • A431 cells were stimulated by different concentrations of EGF for 10 min at 37°C.

Protocols

References

ab126421 has not yet been referenced specifically in any publications.

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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