Recombinant Anti-EGFR antibody [E235] (ab32077)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [E235] to EGFR
- Suitable for: IHC-P, Flow Cyt (Intra), ICC/IF, WB, IP
- Knockout validated
- Reacts with: Mouse, Rat, Human
Related conjugates and formulations
Overview
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Product name
Anti-EGFR antibody [E235]
See all EGFR primary antibodies -
Description
Rabbit monoclonal [E235] to EGFR -
Host species
Rabbit -
Specificity
ab32077 detects Epidermal growth factor receptor (EGFR). It does not cross react with other ERBB family members. This product yielded a strong signal in western blot using A431 (human squamous carcinoma) lysate which naturally overexpresses the EGFR protein. Western blot conditions may need to be optimised for cell lines and tissues that express lower levels of endogenous EGFR. -
Tested applications
Suitable for: IHC-P, Flow Cyt (Intra), ICC/IF, WB, IPmore details -
Species reactivity
Reacts with: Mouse, Rat, Human -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: A431 cell lysate. ICC/IF: A431 cells. Flow Cyt (intra): A431 cells. IHC-P: FFPE mouse skin normal. IP: A431 cell lysate
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General notes
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.
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. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 49% PBS, 50% Glycerol (glycerin, glycerine), 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
E235 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
- Alexa Fluor® 488 Anti-EGFR antibody [E235] (ab205731)
- Alexa Fluor® 647 Anti-EGFR antibody [E235] (ab205732)
- HRP Anti-EGFR antibody [E235] (ab206036)
- PE Anti-EGFR antibody [E235] (ab213332)
- Alexa Fluor® 555 Anti-EGFR antibody [E235] (ab214627)
- Anti-EGFR antibody [E235] - BSA and Azide free (ab227459)
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Compatible Secondaries
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Isotype control
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KO cell lines
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KO cell lysates
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KO cell pellets
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Positive Controls
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Recombinant Protein
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab32077 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 |
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IHC-P | (3) |
1/100 - 1/200. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
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Flow Cyt (Intra) |
1/10 - 1/1000.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
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ICC/IF |
1/500.
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WB |
1/1000 - 1/10000. Detects a band of approximately 180 kDa (predicted molecular weight: 134 kDa).
This product yielded a strong signal in western blot using A431 (human squamous carcinoma) lysate which naturally overexpresses the EGFR protein. Western blot conditions may need to be optimised for cell lines and tissues that express lower levels of endogenous EGFR. |
|
IP |
1/50.
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Notes |
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IHC-P
1/100 - 1/200. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. |
Flow Cyt (Intra)
1/10 - 1/1000. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
ICC/IF
1/500. |
WB
1/1000 - 1/10000. Detects a band of approximately 180 kDa (predicted molecular weight: 134 kDa). This product yielded a strong signal in western blot using A431 (human squamous carcinoma) lysate which naturally overexpresses the EGFR protein. Western blot conditions may need to be optimised for cell lines and tissues that express lower levels of endogenous EGFR. |
IP
1/50. |
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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All lanes : Anti-EGFR antibody [E235] (ab32077) at 1/1000 dilution
Lane 1 : Wild-type HCT 116 cell lysate
Lane 2 : EGFR knockout HCT 116 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : Caco-2 cell lysate
Lysates/proteins at 20 µg per lane.
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 134 kDa
Observed band size: 180 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-EGFR antibody [E235] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32077 was shown to bind specifically to EGFR. A band was observed at 180 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in Egfr knockout cell line ab281597 (knockout cell lysate ab282949). To generate this image, wild-type and Egfr knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [E235] (ab32077)
IHC image of EGFR staining in a formalin fixed, paraffin embedded mouse normal skin tissue section, performed on a Leica Bond™ system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab32077 at 1/200 dilution for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
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All lanes : Anti-EGFR antibody [E235] (ab32077) at 1/1000 dilution
Lane 1 : Caco-2 cell lysate
Lane 2 : A431 cell lysate
Lane 3 : Mouse skin cell lysate
Lane 4 : Rat skin cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 134 kDaThis blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with ab32077 overnight at 4°C in the presence of loading control ab18058 (Mouse monoclonal [SPM227] to Vinculin diluted 1:10000). Antibody binding was detected using IR-labelled goat anti-Rabbit Ab at a 1:10,000 dilution for one hour at room temperature before imaging.
This product yielded a strong signal in western blot using A431 (human squamous carcinoma) lysate which naturally overexpresses the EGFR protein. Western blot conditions may need to be optimised for cell lines and tissues that express lower levels of endogenous EGFR.
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Purified ab32077 at 1/50 dilution (2µg) immunoprecipitating EGFR in A431 whole cell lysate.
Lane 1 (input): A431 (Human epidermoid carcinoma epithelial cell) whole cell lysate 10µg
Lane 2 (+): ab32077 + A431 whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32077 in A431 whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 180 kDa -
Immunocytochemistry/Immunofluorescence analysis of A431 cells labelling EGFR with ab32077 at 1/500. Cells were fixed with 100% methanol. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody.
Control: PBS only.
Nuclear counter stain: DAPI. -
Overlay histogram showing A431 cells stained with ab32077 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32077, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1µg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.
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.
Datasheets and documents
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SDS download
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Datasheet download
References (21)
ab32077 has been referenced in 21 publications.
- Morgan EL et al. E6-mediated activation of JNK drives EGFR signalling to promote proliferation and viral oncoprotein expression in cervical cancer. Cell Death Differ 28:1669-1687 (2021). PubMed: 33303976
- Ticiani E et al. Bisphenol S and Epidermal Growth Factor Receptor Signaling in Human Placental Cytotrophoblasts. Environ Health Perspect 129:27005 (2021). PubMed: 33605785
- Xu Y et al. Virtual Screening of Cablin Patchouli Herb as a Treatment for Heat Stress: A Study Based on Network Pharmacology, Molecular Docking, and Experimental Verification. Evid Based Complement Alternat Med 2021:8057587 (2021). PubMed: 33777163
- Li Y et al. TUC338 Promotes Diffuse Large B Cell Lymphoma Growth via Regulating EGFR/PI3K/AKT Signaling Pathway. J Oncol 2021:5593720 (2021). PubMed: 33986803
- Pan K et al. SPARC promotes pancreatic cancer cell proliferation and migration through autocrine secretion into the extracellular milieu. Oncol Lett 21:485 (2021). PubMed: 33968201