Overview

Properties

Applications

Our Abpromise guarantee covers the use of ab5644 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
ICC/IF 1/100.
WB 1/1000. Detects a band of approximately 185 kDa.

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

  • Ab5644 staining EFGR in 70% confluent log phase A-431 cells treated with 200ng/ml of EGF for 10 minutes by ICC/IF (Immunocytochemistry/Immunofluorescence).  The cells were fixed with 4% paraformaldehyde for 10 minutes; permeabilized with 0.1% Triton X-100 for 10 minutes and blocked with 1% BSA for hour at room temperature. Primary antibody was used at 1/100 dilution. A Goat anti-rabbit IgG (H+L) Superclonal, Alexa Fluor® 488 conjugate was used as the secondary antibody at 1/2000 dilution (image a). Nuclei (image b) were stained with SlowFade® Gold Antifade Mountant with DAPI. F-actin (C) was stained with Rhodamine Phalloidin. Image d represents the merged image showing membrane localization. Image  e represents cells treated with antagonist, Afatinib (1µM for 6hrs) followed by EGF (200 ng/ml for 10 minutes), showing no Phospho-EGFR staining. Panel f shows untreated cells with no signal. Panel g represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

  • Peptide Competition: Extracts prepared from NIH3T3 cells expressing EGFR were starved for 30 hours, then stimulated for 10 minutes with 30 ng/mL EGF (+), or left unstimulated (-), then resolved by SDS-PAGE on a 6% Tris-glycine gel, and transferred to nitrocellulose. Membranes were blocked with a 5% BSA-TBST buffer overnight at 4°C, then were incubated with 0.50 µg/mL ab5644 antibody, following prior incubation with: no peptide (1, 2), the phosphopeptide immunogen (3, 4), or, the non phosphopeptide corresponding to the phosphopeptide immunogen (5, 6). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit   IgG alkaline phosphatase and bands were detected using the Tropix WesternStar method. The data show that only the phosphopeptide corresponding to this site blocks the antibody signal, demonstrating the specificity of the ab5644 antibody for this phosphorylated residue. The data also show the activation of the EGFR after stimulat
  • Western blot analysis using ab5644 shows increased expression of proteins phosphorylated at the tyrosine residues in A-431 and A549 cell lines upon EGF treatment and pre-treatment with EGFR-antagonists, Gefitinib and Afatinib. This results in inhibition of Phospho-EGFR in A-431 and A549 cell lines

  • All lanes : Anti-EGFR (phospho Y1068) antibody (ab5644) at 1/200 dilution

    Lane 1 : A431 whole cell lysate - not treated
    Lane 2 : A431 whole cell lysate - 100ng/ml EGF for 1 minute
    Lane 3 : A431 whole cell lysate - 100ng/ml EGF for 2.5 minutes
    Lane 4 : A431 whole cell lysate - 100ng/ml EGF for 5 minutes
    Lane 5 : A431 whole cell lysate - 100ng/ml EGF for 10 minutes
    Lane 6 : A431 whole cell lysate - 100ng/ml EGF for 20 minutes
    Lane 7 : A431 whole cell lysate - 100ng/ml EGF for 40 minutes

    Secondary
    All lanes : HRP conjugated Goat anti-rabbit antibody

    Developed using the ECL technique.

    Performed under reducing conditions.

    Exposure time: 2 minutes

    See Abreview

References

This product has been referenced in:
  • Wasén C  et al. Epidermal growth factor receptor function in the human urothelium. Int Urol Nephrol 50:647-656 (2018). WB, IHC-P . Read more (PubMed: 29508172) »
  • Ali A  et al. Fatty acid synthase mediates EGFR palmitoylation in EGFR mutated non-small cell lung cancer. EMBO Mol Med N/A:N/A (2018). Human . Read more (PubMed: 29449326) »
See all 17 Publications for this product

Customer reviews and Q&As

1-7 of 7 Abreviews or Q&A

Application
Western blot
Loading amount
50 µg
Gel Running Conditions
Reduced Denaturing
Sample
Mouse Tissue lysate - whole (heart)
Specification
heart
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Ms. sarah shen

Verified customer

Submitted May 12 2014

Answer

Thank you for contacting us.

To our knowledge, ab5644 has not been tested in immunohistochemistry (paraffin) however, this does not mean that it will not work in this application it just means that we have not tested it ourselves. By participating in our AbTrial program you can now use ab5644 in immunohistochemistry (paraffin) without financial risk.

Simply follow these easy steps below to apply for our AbTrial Program:

1. Reply to this email, letting us know you are interested in testing this product in immunohistochemistry (paraffin).

2. Our scientists will email you an inactive personal discount code for the value of the product.

3. Purchase and test the product at the regular price.

4. Submit your results, including your discount code in the additional notes section of your Abreview.

5. Once the Abreview is submitted, whether positive or negative, the discount code will become active.

6. Apply your discount code on your next order to receive that value off.

Please let me know if you have any questions about this offer and I would be happy to help you further.

The Terms and Conditions of this offer can be found at: www.abcam.com/abtrial.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Rat Tissue lysate - whole (Liver)
Loading amount
30 µg
Specification
Liver
Gel Running Conditions
Reduced Denaturing (12)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Jun 25 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Tissue lysate - whole (Liver)
Loading amount
50 µg
Specification
Liver
Gel Running Conditions
Reduced Denaturing (12)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

Abcam user community

Verified customer

Submitted May 02 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Rat Tissue lysate - whole (Liver)
Loading amount
50 µg
Specification
Liver
Gel Running Conditions
Reduced Denaturing (12)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Jun 08 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (Lung)
Loading amount
70 µg
Specification
Lung
Treatment
EGF 100ng/ml 2.5 minutes
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5%

Dr. Samir Nuseibeh

Verified customer

Submitted Oct 23 2006

Answer

Thank you for your enquiry. I am sorry to hear that you have been having difficulties with this antibody. I have been in touch with the originators of ab5638, ab5644 and ab14017 with regards the band sizes that you have detected. I have received the following comments. The mentioned that the most likely explanation for the bands that you have been detecting and the mass of protein that you have been loading is that the the cell preparation that you have been using does not contain any EGFR phosphorylated at these 2 specific sites (ab5638 and ab5644). When the correct target does not exist (or exists in concentrations too low for the antibody to detect), the antibody will bind to either a lower affinity phospho. protein or to the most concentrated protein in the lysate: Neither bindings are specific. The originator also recommended that you try titrating the extracts themselves and the secondary antibody to determine whether the target protein is indeed the protein detected by the antibody or a non-specific band detected by the secondary antiserum. I would like to recommend that you stimulate cells using EGF to induce phosphorylation as shown in the western blot on the datasheet of ab5638 where EGF induced phosphorylation of EGFR. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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