Anti-EGFR (phospho Y1068) antibody (ab5644)
Key features and details
- Rabbit polyclonal to EGFR (phospho Y1068)
- Suitable for: ICC/IF, WB
- Reacts with: Human
- Isotype: IgG
Overview
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Product name
Anti-EGFR (phospho Y1068) antibody
See all EGFR primary antibodies -
Description
Rabbit polyclonal to EGFR (phospho Y1068) -
Host species
Rabbit -
Tested Applications & Species
See all applications and species dataApplication Species ICC/IF HumanWB Human
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Immunogen
Synthetic phosphopeptide (Human) derived from the region of EGFR that contains tyrosine 1068.
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Positive control
- Purchase matching WB positive control:Recombinant human EGFR protein
- WB: A-431 and A549 whole cell lysate treated with EGF (200 ng/mL for 10 minutes). ICC: A-431 cells treated with 200 ng/mL of EGF for 10 minutes.
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General notes
The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing the problem with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation.
One factor contributing to the crisis is the use of antibodies that are not suitable. This can lead to misleading results and the use of incorrect data informing project assumptions and direction. To help address this challenge, we have introduced an application and species grid on our primary antibody datasheets to make it easy to simplify identification of the right antibody for your needs.
Learn more here.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles. -
Storage buffer
pH: 7.30
Preservative: 0.05% Sodium azide
Constituents: PBS, 50% Glycerol, 0.1% BSA -
Concentration information loading... -
Purity
Immunogen affinity purified -
Purification notes
The 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. -
Clonality
Polyclonal -
Isotype
IgG -
Research areas
Associated products
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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 ab5644 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 |
|---|---|
| ICC/IF |
Human
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| WB |
Human
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| Application | Abreviews | Notes |
|---|---|---|
| ICC/IF |
1/100.
|
|
| WB | (5) |
1/1000. Detects a band of approximately 185 kDa.
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| Notes |
|---|
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ICC/IF
1/100. |
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WB
1/1000. Detects a band of approximately 185 kDa. |
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
- Omim: 131550 Human
- SwissProt: P00533 Human
- Unigene: 488293 Human
- Unigene: 605083 Human
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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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Immunocytochemistry/ Immunofluorescence - Anti-EGFR (phospho Y1068) antibody (ab5644)
Immunofluorescence analysis of A431 (Human epidermoid carcinoma cell line) cells labeling EGFR with ab5644 at 1/100 dilution, followed by Goat anti-rabbit IgG (H+L) Superclonal, Alexa Fluor® 488 conjugate was used as the secondary antibody at 1/2000 dilution (Panel a). Nuclei (Panel b) were stained with SlowFade® Gold Antifade Mountant with DAPI. F-actin (Panel c) was stained with Rhodamine Phalloidin. Panel (d) represents the merged image showing membrane localization. Panel (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 cells (in 70% confluent log phase treated with 200ng/ml of EGF for 10 minutes) 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. The images were captured at 60X magnification.
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Western blot - Anti-EGFR (phospho Y1068) antibody (ab5644)All lanes : Anti-EGFR (phospho Y1068) antibody (ab5644) at 1/1000 dilution
Lane 1 : A431 (Human epidermoid carcinoma cell line) whole cell lysate with skimmed milk
Lane 2 : A431 whole cell lysate treated with EGF (200 ng/mL for 10 minutes) with skimmed milk
Lane 3 : A431 whole cell lysate treated with Gefitinib followed by EGF (1uM for 16 hours, 200 ng/mL for 10 minutes) with skimmed milk
Lane 4 : A431 whole cell lysate treated with Afatinib followed by EGF (0.5 uM for 6 hours, 200 ng/mL for 10 minutes) with skimmed milk
Lane 5 : A549 (Human lung carcinoma cell line) whole cell lysate with skimmed milk
Lane 6 : A549 whole cell lysate treated with EGF (200 ng/mL for 10 minutes) with skimmed milk
Lane 7 : A549 whole cell lysate treated with Afatinib followed by EGF (0.5 uM for 6 hours, 200 ng/mL for 10 minutes) with skimmed milk
Lysates/proteins at 30 µg per lane.
Blocking peptides at 5 % per lane.
Secondary
All lanes : Goat anti-Rabbit IgG Superclonal Secondary Antibody, HRP at 1/4000 dilutionWestern 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
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Western blot - Anti-EGFR (phospho Y1068) antibody (ab5644)This image is courtesy of an Abreview submitted by Mr Samir NuseibehAll lanes : Anti-EGFR (phospho Y1068) antibody (ab5644) at 1/200 dilution
Lane 1 : A431 (Human epidermoid carcinoma cell line) 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
References (35)
ab5644 has been referenced in 35 publications.
- Nakano Y et al. Clinical importance of long non-coding RNA LINC00460 expression in EGFR-mutant lung adenocarcinoma. Int J Oncol 56:243-257 (2020). PubMed: 31789388
- Song X et al. Zi Shen Huo Luo Formula Enhances the Therapeutic Effects of Angiotensin-Converting Enzyme Inhibitors on Hypertensive Left Ventricular Hypertrophy by Interfering With Aldosterone Breakthrough and Affecting Caveolin-1/Mineralocorticoid Receptor Colocalization and Downstream Extracellular Signal-Regulated Kinase Signaling. Front Pharmacol 11:383 (2020). PubMed: 32317965
- Elkhider A et al. Aquaporin 5 promotes tumor migration and angiogenesis in non-small cell lung cancer cell line H1299. Oncol Lett 19:1665-1672 (2020). PubMed: 32194658
- 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
- Wang D & Bao B Gallic Acid Impedes Non-Small Cell Lung Cancer Progression via Suppression of EGFR-Dependent CARM1-PELP1 Complex. Drug Des Devel Ther 14:1583-1592 (2020). PubMed: 32425504
