Product nameAnti-EGFR (phospho Y1068) antibody
See all EGFR primary antibodies
DescriptionRabbit polyclonal to EGFR (phospho Y1068)
Tested applicationsSuitable for: ICC/IF, WBmore details
Species reactivityReacts with: Rat, Human
Predicted to work with: Mouse, Chicken
Synthetic phosphopeptide (Human) derived from the region of EGFR that contains tyrosine 1068.
- Purchase matching WB positive control:Recombinant human EGFR protein
- NIH3T3 cells expressing human EGFR, A431 cells +/- EGF.
Storage instructionsShipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
Storage bufferpH: 7.30
Preservative: 0.05% Sodium azide
Constituents: PBS, 50% Glycerol, 0.1% BSA
Concentration information loading...
PurityImmunogen affinity purified
Purification notesThe antibody has been negatively preadsorbed using (i) a non phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated epidermal growth factor receptor (EGFR), and (ii) a generic tyrosine phosphorylated peptide to remove antibody that is reactive with phosphotyrosine, irrespective of the sequence. The final product is generated by affinity chromatography using an EGFR-derived peptide that is phosphorylated at tyrosine 1068.
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.
|WB||1/1000. Detects a band of approximately 185 kDa.|
FunctionReceptor 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 specificityUbiquitously expressed. Isoform 2 is also expressed in ovarian cancers.
Involvement in diseaseLung cancer
Inflammatory skin and bowel disease, neonatal, 2
Sequence similaritiesBelongs to the protein kinase superfamily. Tyr protein kinase family. EGF receptor subfamily.
Contains 1 protein kinase domain.
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 localizationSecreted 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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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
All lanes : HRP conjugated Goat anti-rabbit antibody
Developed using the ECL technique.
Performed under reducing conditions.
Exposure time: 2 minutes
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) »