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    egfr-antibody-e235-ab32077.pdf

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Signal Transduction Protein Phosphorylation Tyrosine Kinases Receptor Tyrosine Kinases
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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-EGFR antibody [E235] (ab32077)

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  • SDS
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Western blot - Anti-EGFR antibody [E235] (ab32077)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [E235] (ab32077)
  • Western blot - Anti-EGFR antibody [E235] (ab32077)
  • Immunoprecipitation - Anti-EGFR antibody [E235] (ab32077)
  • Immunocytochemistry/ Immunofluorescence - Anti-EGFR antibody [E235] (ab32077)
  • Flow Cytometry (Intracellular) - Anti-EGFR antibody [E235] (ab32077)
  • 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

Conjugates logo Related conjugates and formulations

Alexa Fluor® 488 Alexa Fluor® 555 Alexa Fluor® 647 Carrier Free HRP PE

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Recombinant human EGFR protein (Active) (ab155639)
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Anti-EGFR antibody [EP38Y] - Low endotoxin, Azide free (ab174481)
Knockout
Product image
Human EGFR knockout HeLa cell line (ab255385)

View more associated products

Overview

  • 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.

  • 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
  • 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
    For more information see here.

    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

  • 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...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    E235
  • Isotype

    IgG
  • Research areas

    • Signal Transduction
    • Protein Phosphorylation
    • Tyrosine Kinases
    • Receptor Tyrosine Kinases
    • Signal Transduction
    • Growth Factors/Hormones
    • EGF
    • Cell Biology
    • Cell Cycle
    • Cell differentiation
    • Microbiology
    • Organism
    • Virus
    • RNA Virus
    • ssRNA positive strand virus
    • SARS Coronavirus
    • Cancer
    • Growth factors
    • EGF
    • Cancer
    • Signal transduction
    • Protein phosphorylation
    • Tyrosine kinases
    • Receptor tyrosine kinases
    • Cancer
    • Oncoproteins/suppressors
    • Oncoproteins
    • Growth factor receptors
    • Cancer
    • Tumor biomarkers
    • Receptors
    • Neuroscience
    • Development
    • Neuroscience
    • Processes

Associated products

  • 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)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed (ab96899)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • KO cell lines

    • Human EGFR knockout HeLa cell line (ab255385)
  • KO cell lysates

    • Human EGFR knockout HeLa cell lysate (ab263845)
  • KO cell pellets

    • Human EGFR knockout HeLa cell pellet (ab278888)
  • Positive Controls

    • A-431 whole cell lysate (ab7909)
  • Recombinant Protein

    • Recombinant human EGFR protein (Active) (ab155639)
  • Related Products

    • Human EGFR ELISA Kit (ERBB) (ab100505)

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
IHC-P (3)
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.
Notes
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

  • 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).
  • Target information above from: UniProt accession P00533 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • 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
    • Unigene: 420648 Mouse
    • Unigene: 439882 Mouse
    • Unigene: 8534 Mouse
    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
    • 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

  • Western blot - Anti-EGFR antibody [E235] (ab32077)
    Western blot - Anti-EGFR antibody [E235] (ab32077)
    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.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [E235] (ab32077)
    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.

  • Western blot - Anti-EGFR antibody [E235] (ab32077)
    Western blot - Anti-EGFR antibody [E235] (ab32077)
    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 kDa



    This 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.

  • Immunoprecipitation - Anti-EGFR antibody [E235] (ab32077)
    Immunoprecipitation - Anti-EGFR antibody [E235] (ab32077)

    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 - Anti-EGFR antibody [E235] (ab32077)
    Immunocytochemistry/ Immunofluorescence - Anti-EGFR antibody [E235] (ab32077)

    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.

  • Flow Cytometry (Intracellular) - Anti-EGFR antibody [E235] (ab32077)
    Flow Cytometry (Intracellular) - Anti-EGFR antibody [E235] (ab32077)

    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. 

     

     

  • Anti-EGFR antibody [E235] (ab32077)
    Anti-EGFR antibody [E235] (ab32077)

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.

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (21)

Publishing research using ab32077? Please let us know so that we can cite the reference in this datasheet.

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
View all Publications for this product

Customer reviews and Q&As

Show All Reviews Q&A
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1-3 of 3 Abreviews or Q&A

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-EGFR antibody [E235]

Inconclusive
Abreviews
Abreviews
abreview image
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Rat Tissue sections (Developing skin)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citric a cid
Permeabilization
No
Specification
Developing skin
Blocking step
BSA as blocking agent for 10 minute(s) · Concentration: 2% · Temperature: 21°C
Fixative
Formaldehyde
Read More
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

MR. Carl Hobbs

Verified customer

Submitted Feb 17 2018

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-EGFR antibody [E235]

Inconclusive
Abreviews
Abreviews
abreview image
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Skin)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citric acid
Permeabilization
No
Specification
Skin
Blocking step
BSA as blocking agent for 10 minute(s) · Concentration: 2% · Temperature: 21°C
Fixative
Formaldehyde
Read More
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

MR. Carl Hobbs

Verified customer

Submitted Feb 16 2018

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-EGFR antibody [E235]

Excellent
Abreviews
Abreviews
abreview image
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Placenta)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citric acid
Permeabilization
No
Specification
Placenta
Blocking step
BSA as blocking agent for 10 minute(s) · Concentration: 2% · Temperature: 21°C
Fixative
Formaldehyde
Read More
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

MR. Carl Hobbs

Verified customer

Submitted Jan 19 2018

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