Recombinant Anti-EGFR (phospho Y1068) antibody [EP774Y] (ab40815)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EP774Y] to EGFR (phospho Y1068)
- Suitable for: Dot blot, WB, ICC/IF, IHC-P, Flow Cyt
- Reacts with: Rat, Human, Respiratory syncytial virus
Overview
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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.
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Tested Applications & Species
Application Species Flow Cyt HumanICC/IF HumanRespiratory syncytial virusIHC-P HumanWB Human -
Immunogen
Synthetic peptide within Human EGFR (phospho Y1068). The exact sequence is proprietary.
(Peptide available asab190788) -
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.
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General notes
An immunizing peptide is available for this antibody as ab190788.
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
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.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
Dissociation constant (KD)
KD = 3.60 x 10 -11 M Learn more about KD -
Storage buffer
pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol, 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EP774Y -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Isotype control
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Recombinant Protein
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Related Products
Applications
The Abpromise guarantee
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.
Tested applications are guaranteed to work and covered by our Abpromise guarantee.
Predicted to work for this combination of applications and species but not guaranteed.
Does not work for this combination of applications and species.
Application | Species |
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Flow Cyt |
Human
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ICC/IF |
Human
Respiratory syncytial virus
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IHC-P |
Human
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WB |
Human
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All applications |
Mouse
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Application | Abreviews | Notes |
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Dot blot |
1/1000.
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WB | (2) |
1/500 - 1/5000. Predicted molecular weight: 170 kDa.
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ICC/IF |
1/250 - 1/500.
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IHC-P | (11) |
1/250 - 1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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.
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Notes |
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Dot blot
1/1000. |
WB
1/500 - 1/5000. Predicted molecular weight: 170 kDa. |
ICC/IF
1/250 - 1/500. |
IHC-P
1/250 - 1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. 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
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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
modificationsPhosphorylation 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
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Database links
- Entrez Gene: 1956 Human
- Entrez Gene: 13649 Mouse
- Entrez Gene: 24329 Rat
- Omim: 131550 Human
- SwissProt: P00533 Human
- SwissProt: Q01279 Mouse
- Unigene: 488293 Human
- Unigene: 605083 Human
see all -
Alternative names
- Avian erythroblastic leukemia viral (v erb b) oncogene homolog antibody
- Cell growth inhibiting protein 40 antibody
- Cell proliferation inducing protein 61 antibody
see all
Images
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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).
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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.
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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.
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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 kDaThis antibody detects high background.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR (phospho Y1068) antibody [EP774Y] (ab40815)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.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR (phospho Y1068) antibody [EP774Y] (ab40815)Wodziak D et al., PLoS One, 11, e0164968, 2016 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/
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).
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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).
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR (phospho Y1068) antibody [EP774Y] (ab40815)This image is courtesy of an Abreview submitted by Mr. Carl Hobbs.
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.
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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.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR (phospho Y1068) antibody [EP774Y] (ab40815)
Immunohistochemical staining of untreated (A) and Phosphatase- treated (B) paraffin-embedded breast adenocarcinoma tissue using ab40815 (unpurified).
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Immunofluorescent staining of untreated (C) and Phosphatase- treated (D) A431 cells using ab40815 (unpurified).
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR (phospho Y1068) antibody [EP774Y] (ab40815)
ab40815 (unpurified) showing positive staining in Papillary carcinoma of thyroid gland tissue.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR (phospho Y1068) antibody [EP774Y] (ab40815)
ab40815 (unpurified) showing positive staining in Glioma tissue.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR (phospho Y1068) antibody [EP774Y] (ab40815)
ab40815 (unpurified) showing positive staining in Cervical carcinoma tissue.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR (phospho Y1068) antibody [EP774Y] (ab40815)This image is submitted courtesy of an anonymous Abreview.
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).
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
References (82)
ab40815 has been referenced in 82 publications.
- Chen L et al. EGFR inhibitor AG1478 blocks the formation of 3D structures mainly through ERK signaling pathway in Matrigel-induced 3D reconstruction of eccrine sweat gland-like structures. J Mol Histol 51:191-197 (2020). PubMed: 32219645
- Deng QF et al. Cyclooxygenase-2 mediates gefitinib resistance in non-small cell lung cancer through the EGFR/PI3K/AKT axis. J Cancer 11:3667-3674 (2020). PubMed: 32284763
- Xu Q et al. Association of iRhom1 and iRhom2 expression with prognosis in patients with cervical cancer and possible signaling pathways. Oncol Rep 43:41-54 (2020). PubMed: 31661139
- Pereira PMR et al. HER2-Targeted PET Imaging and Therapy of Hyaluronan-Masked HER2-Overexpressing Breast Cancer. Mol Pharm 17:327-337 (2020). PubMed: 31804840
- Šuštic T et al. RUNX2/CBFB modulates the response to MEK inhibitors through activation of receptor tyrosine kinases in KRAS-mutant colorectal cancer. Transl Oncol 13:201-211 (2020). PubMed: 31865182