Recombinant
RabMAb

Recombinant Anti-EGFR antibody [E234] (ab32198)

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Overview

  • Product name

    Anti-EGFR antibody [E234]
    See all EGFR primary antibodies

  • Description

    Rabbit monoclonal [E234] to EGFR

  • Host species

    Rabbit

  • Specificity

    ab32198 detects Epidermal growth factor receptor (EGFR). It does not cross react with other ERBB family members. This product yielded a strong signal in western blot using A431 (human squamous carcinoma) lysate which naturally overexpresses the EGFR protein. Western blot conditions may need to be optimised for cell lines and tissues that express lower levels of endogenous EGFR.

  • Tested applications

    Suitable for: WB, Flow Cyt, ICC/IF, IHC-Pmore details

  • Species reactivity

    Reacts with: Human
    Predicted to work with: Cow, Pig

  • Immunogen

    Synthetic peptide corresponding to Human EGFR aa 1-100 (N terminal).
    Database link: P00533

  • Positive control

    • WB: A431 cell lysate. IHC-P: FFPE human skin tissue sections. ICC/IF: A431 cells.

  • General notes

    Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

     

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

Properties

Applications

Our Abpromise guarantee covers the use of ab32198 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
WB 1/1000. Detects a band of approximately 134 kDa (predicted molecular weight: 180 kDa).

For unpurified use at 1/500.

This product yielded a strong signal in western blot using A431 (human squamous carcinoma) lysate which naturally overexpresses the EGFR protein. Western blot conditions may need to be optimised for cell lines and tissues that express lower levels of endogenous EGFR.
Flow Cyt 1/200.

For unpurified use at 1/50.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
ICC/IF 1/100.
IHC-P 1/100. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

Target

  • 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

  • Database links

    • Alternative names

      • Avian erythroblastic leukemia viral (v erb b) oncogene homolog antibody
      • Cell growth inhibiting protein 40 antibody
      • Cell proliferation inducing protein 61 antibody
      • EGF R antibody
      • EGFR antibody
      • EGFR_HUMAN antibody
      • Epidermal growth factor receptor (avian erythroblastic leukemia viral (v erb b) oncogene homolog) antibody
      • Epidermal growth factor receptor (erythroblastic leukemia viral (v erb b) oncogene homolog avian) antibody
      • Epidermal growth factor receptor antibody
      • erb-b2 receptor tyrosine kinase 1 antibody
      • ERBB antibody
      • ERBB1 antibody
      • Errp antibody
      • HER1 antibody
      • mENA antibody
      • NISBD2 antibody
      • Oncogen ERBB antibody
      • PIG61 antibody
      • Proto-oncogene c-ErbB-1 antibody
      • Receptor tyrosine protein kinase ErbB 1 antibody
      • Receptor tyrosine-protein kinase ErbB-1 antibody
      • SA7 antibody
      • Species antigen 7 antibody
      • Urogastrone antibody
      • v-erb-b Avian erythroblastic leukemia viral oncogen homolog antibody
      • wa2 antibody
      • Wa5 antibody
      see all

    Images

    • Immunocytochemistry/ Immunofluorescence - Anti-EGFR antibody [E234] (ab32198)
      Immunocytochemistry/ Immunofluorescence - Anti-EGFR antibody [E234] (ab32198)

      ab32198 (unpurified) stained A431 cells. The cells were 100% methanol fixed for 10 minutes at -20°C and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab32198 at 1in100 dilution) overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [E234] (ab32198)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [E234] (ab32198)

      IHC image of EGFR staining in human skin formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab32198 (unpurified) at 1/100 dilution for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

       

      For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

      *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

    References

    This product has been referenced in:

    • Gireud-Goss M  et al. Distinct mechanisms enable inward or outward budding from late endosomes/multivesicular bodies. Exp Cell Res 372:1-15 (2018). Read more (PubMed: 30144444) »
    See 1 Publication for this product

    Publishing research using ab32198? Please let us know so that we can cite the reference in this datasheet.

    Customer reviews and Q&As

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    Western blot abreview for Anti-EGFR antibody [E234]

     Inconclusive
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Bos taurus Tissue lysate - whole (Liver)
    Gel Running Conditions
    Reduced Denaturing
    Loading amount
    100 µg
    Specification
    Liver
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
    Read More

    Mr. Christian Beauvallet

    Verified customer

    Submitted Mar 20 2018

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