Overview

  • Product name
    Anti-EGFR (phospho Y1068) antibody [EP774Y]
    See all EGFR primary antibodies
  • Description
    Rabbit monoclonal [EP774Y] to EGFR (phospho Y1068)
  • Host species
    Rabbit
  • Specificity

    Recognises EGFR phosphorylated on Tyrosine 1068 of the mature human isoform 1 (corresponding to Y1092 from the precursor form P00533-1/p170)

    The mouse recommendation is based on the WB results. We do not guarantee IHC-P for mouse.

  • Tested applications
    Suitable for: Dot blot, IHC-FoFr, WB, ICC/IF, IHC-P, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide within Human EGFR (phospho Y1068). The exact sequence is proprietary.
    (Peptide available as ab190788)

  • Positive control
    • Purchase matching WB positive control:Recombinant human EGFR protein
    • IHC-P: Human breast adenocarcinoma, papillary carcinoma of thyroid glad, glioma, cervical carcinoma and prostate cancer tissue; Mouse E17 embryo head tissue and mouse pancreas tissue. ICC/IF: A431 cells and WD-PBEC cultures. WB: A431 cells and A431 cells treated with EGF. Dot Blot: EGFR (pY1068) peptide.
  • General notes

    An immunizing peptide is available for this antibody as ab190788.

    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 rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab40815 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
Dot blot 1/1000.
IHC-FoFr Use at an assay dependent concentration. PubMed: 18948956
WB 1/500 - 1/5000. Predicted molecular weight: 170 kDa.Can be blocked with EGFR (phospho Y1068) peptide (ab190788).
ICC/IF 1/250 - 1/500.
IHC-P 1/250 - 1/500.

See IHC antigen retrieval protocols.

The mouse recommendation is based on the WB results. We do not guarantee IHC-P for mouse.

Flow Cyt 1/800.

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

  • Flow Cytometry analysis of A431 (Human epidermoid carcinoma epithelial cell) treated with 200 ng/ml EGF for 15 minutes cells labeling EGFR with purified ab40815 at 1:800 dilution (1 µg/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1:2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue). Untreated A431 cells (Green).
  • All lanes : Anti-EGFR (phospho Y1068) antibody [EP774Y] (ab40815) at 1/1000 dilution (Purified)

    Lane 1 : A431 (Human epidermoid carcinoma epithelial cell) whole cell lysates
    Lane 2 : A431 (Human epidermoid carcinoma epithelial cell) treated with 10 ng/ml EGF for 30 minutes whole cell lysates
    Lane 3 : A431 (Human epidermoid carcinoma epithelial cell) treated with 10 ng/ml EGF for 30 minutes. Then the membrane was incubated with phosphatase

    Lysates/proteins at 15 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 170 kDa
    Observed band size: 175 kDa
    why is the actual band size different from the predicted?



    This antibody detects high background.

  • Immunocytochemistry/ Immunofluorescence analysis of A431 (Human epidermoid carcinoma epithelial cell) treated with 100 ng/ml EGF for 10 minutes cells labeling EGFR with purified ab40815 at 1:500 dilution (1.8 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) at 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.
  • All lanes : Anti-EGFR (phospho Y1068) antibody [EP774Y] (ab40815) at 1/1000 dilution (purified)

    Lane 1 : A431 (Human epidermoid carcinoma epithelial cell) whole cell lysates
    Lane 2 : A431 (Human epidermoid carcinoma epithelial cell) treated with 10 ng/ml EGF for 30 minutes whole cell lysates
    Lane 3 : A431 (Human epidermoid carcinoma epithelial cell) treated with 10 ng/ml EGF for 30 minutes. Then the membrane was incubated with phosphatase

    Lysates/proteins at 15 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 170 kDa



    This antibody detects high background.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human breast cancer tissue sections labeling EGFR with purified ab40815 at 1:500 dilution (1.75 µg/ml). Heat mediated antigen retrieval was performed using heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.
  • Total EGFR (green) subcellular localization with and without caerulein-induced pancreatitis as determined by immunohistochemistry and confocal imaging. The nuclei were identified with DAPI stain (blue). Scale bars = 50 μm. Calreticulin and E-cadherin (red) served as markers for the endoplasmic reticulum and plasma membrane, respectively, and were used to quantify EGFR subcellular location. Pancreatitis was induced with the 1-day protocol of 8 hourly caerulein injections in 6–8 week old wild-type (wt) and 3-week old AGR2-/- (ko) mice.

    For immunofluorescence, antigen retrieval was performed in a pressure cooker set to 118°C. The slides were incubated in antigen unmasking solution (DAKO) for 3 min followed by equilibration at room temperature for 1 hr. The slides were then placed in 5% serum blocking solution (goat, horse, or rabbit serum as appropriate) for 30 min to block nonspecific binding of antibody to the tissue. The sections were incubated with primary antibody diluted in 2% serum overnight at 4°C. The respective secondary antibodies were used at predetermined dilutions. Immunofluorescence slides were mounted with media containing DAPI stain (Vectashield, Vector Laboratories).

  • WD-PBEC cultures were infected with RSV clinical isolate BT2a and stained for EGFR (red) and RSV F (RSV F, green) expression.

    For WD-PBECs, pediatric bronchial epithelial cells (PBEC) were obtained, via written parental consent, from bronchial brushings of children undergoing elective surgery at the Royal Belfast Hospital for Sick Children, and the procedures were approved by the Office of Research Ethics Committees Northern Ireland. PBEC were expanded in collagen-coated flasks using airway epithelial cell media and supplements (Lonza), then seeded onto transwell inserts (Corning), and then air-liquid interface (ALI) cultures were initiated and maintained 21 days in order to establish well-differentiated (WD)-PBECs. Paraformaldehyde-fixed and permeabilized WD-PBEC were stained for RSV F protein expression and were stained with anti-phospho-(p)-EGFR (Abcam, ab40815, unpurified). WD-PBEC cultures were infected with RSV subgroup A clinical isolate BT2a. Fluorescent images were obtained with a SP5 confocal DMI 6000 inverted microscope (Leica).

  • Formaldehyde-fixed, paraffin-embedded mouse e17 embryo head (Developing tooth) tissue stained for EGFR (phospho Y1068) using ab40815 (unpurified) at 1/1000 dilution in immunohistochemical analysis, followed by Goat anti Rabbit IgG (Biotin) at 1/300 dilution.

    See Abreview

  • Dot blot analysis of EGFR (pY1068) peptide (Lane 1), SMAD5 (unmodified) peptide labelling EGFR (pY1068) with ab40815 (unpurified) at a dilution of 1/1000. Peroxidase conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody at a dilution of 1/2500.

    Blocking and dilution buffer: 5% NFDM/TBST.

    Exposure time: 3 minutes.

  • Immunohistochemical staining of untreated (A) and Phosphatase- treated (B) paraffin-embedded breast adenocarcinoma tissue using ab40815 (unpurified).

  • Immunofluorescent staining of untreated (C) and Phosphatase- treated (D) A431 cells using ab40815 (unpurified).

  • ab40815 (unpurified) showing positive staining in Papillary carcinoma of thyroid gland tissue.

  • ab40815 (unpurified) showing positive staining in Glioma tissue.

  • ab40815 (unpurified) showing positive staining in Cervical carcinoma tissue.

  • Equilibrium disassociation constant (KD)
    Learn more about KD

    Click here to learn more about KD
  • Formaldehyde-fixed, paraffin-embedded human prostate cancer tissue stained for EGFR (phospho Y1068) using ab40815 (unpurified) at 1/200 dilution in immunohistochemical analysis, followed by Goat anti Rabbit IgG (Biotin).

    See Abreview

References

This product has been referenced in:
  • Liu CY  et al. Varlitinib Downregulates HER/ERK Signaling and Induces Apoptosis in Triple Negative Breast Cancer Cells. Cancers (Basel) 11:N/A (2019). Read more (PubMed: 30658422) »
  • Naito K  et al. Effect of selective serotonin (5-HT)2B receptor agonist BW723C86 on epidermal growth factor/transforming growth factor-a receptor tyrosine kinase and ribosomal p70 S6 kinase activities in primary cultures of adult rat hepatocytes. Biol Pharm Bull N/A:N/A (2019). Read more (PubMed: 30713268) »
See all 57 Publications for this product

Customer reviews and Q&As

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1-10 of 12 Abreviews

Application
Western blot
Sample
Human Tissue lysate - whole (stomach cancer tissue)
Loading amount
30 µg
Specification
stomach cancer tissue
Treatment
10uM cannabinoid antagonist
Gel Running Conditions
Reduced Denaturing (10% SDS-PAGE)
Blocking step
Milk as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Apr 08 2013

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Intestine)
Specification
Intestine
Fixative
Paraformaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate pH6
Permeabilization
Yes - 0.1% Tx100 in PBS
Blocking step
Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Mar 20 2012

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (skin of the ear)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Tris-EDTA pH=8
Permeabilization
Yes - 0.05% tween20
Specification
skin of the ear
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: RT°C
Fixative
Formaldehyde

Dr. Juan Fons

Verified customer

Submitted Mar 27 2019

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Placenta and TR146 cell line)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citric acid
Permeabilization
No
Specification
Placenta and TR146 cell line
Blocking step
BSA as blocking agent for 10 minute(s) · Concentration: 2% · Temperature: 21°C
Fixative
Formaldehyde

Mr. Carl Hobbs

Verified customer

Submitted Oct 04 2018

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (human lung cancer)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: citrate ph 6
Permeabilization
No
Specification
human lung cancer
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted May 09 2018

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Rat Tissue sections (Developing cartilage)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citric acid
Permeabilization
No
Specification
Developing cartilage
Blocking step
BSA as blocking agent for 10 minute(s) · Concentration: 2% · Temperature: 21°C
Fixative
Formaldehyde

Mr. Carl Hobbs

Verified customer

Submitted Feb 17 2018

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Developing tooth)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citric acid
Permeabilization
No
Specification
Developing tooth
Blocking step
BSA as blocking agent for 10 minute(s) · Concentration: 2% · Temperature: 21°C
Fixative
Formaldehyde

Mr. Carl Hobbs

Verified customer

Submitted Feb 17 2018

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Prostate cancer)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate pH6.0
Permeabilization
No
Specification
Prostate cancer
Blocking step
Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Aug 02 2016

Application
Immunohistochemistry (Frozen sections)
Sample
Human Cell (Skin)
Permeabilization
No
Specification
Skin
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted May 27 2016

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Tumor)
Specification
Tumor
Fixative
Paraformaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: in cell conditioning solution for 36 minutes (CC1 Tris-based buffer pH 8.4) using a standard protocol
Permeabilization
No

Mrs. Surekha Pimple

Verified customer

Submitted Mar 09 2012

1-10 of 12 Abreviews

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