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Signal Transduction Protein Phosphorylation Tyrosine Kinases Receptor Tyrosine Kinases
使用敲除细胞株进行验证重组RabMAb

重组Anti-EGFR抗体[E235] (ab32077)

  • Datasheet
  • SDS
Reviews (3) Submit a question References (10)

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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)
  • Western blot - Anti-EGFR antibody [E235] (ab32077)
  • Immunocytochemistry/ Immunofluorescence - Anti-EGFR antibody [E235] (ab32077)
  • Flow Cytometry - 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, ICC/IF, WB, Flow Cyt, IP
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

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敲除
Product image
Human EGFR knockout HeLa cell line (ab255385)

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概述

  • 产品名称

    Anti-EGFR抗体[E235]
    参阅全部 EGFR 一抗
  • 描述

    兔单克隆抗体[E235] to EGFR
  • 宿主

    Rabbit
  • 特异性

    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 & Species

    Application Species
    Flow Cyt
    Human
    ICC/IF
    Human
    IHC-P
    Mouse
    IP
    Human
    WB
    Human
    See all applications and species data
  • 免疫原

    Synthetic peptide within Human EGFR aa 1150 to the C-terminus (C terminal). The exact sequence is proprietary.

  • 阳性对照

    • WB: A431 cell lysate. ICC/IF: A431 cells. Flow Cyt: A431 cells. IHC-P: FFPE mouse skin normal. IP: A431 cell lysate
  • 常规说明

    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.

    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.

性能

  • 形式

    Liquid
  • 存放说明

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
  • 存储溶液

    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 49% PBS, 50% Glycerol (glycerin, glycerine), 0.05% BSA
  • Concentration information loading...
  • 纯度

    Protein A purified
  • 克隆

    单克隆
  • 克隆编号

    E235
  • 同种型

    IgG
  • 研究领域

    • Signal Transduction
    • Protein Phosphorylation
    • Tyrosine Kinases
    • Receptor Tyrosine Kinases
    • Signal Transduction
    • Growth Factors/Hormones
    • EGF
    • Cell Biology
    • Cell Cycle
    • Cell differentiation
    • 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

相关产品

  • Affibody® Molecule

    • Anti-EGFR Affibody® Molecule Agarose Immobilized (ab36039)
  • 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)
  • Related Products

    • Human EGFR ELISA Kit (ERBB) (ab100505)
    • Human soluble EGFR ELISA Kit (ab193764)

应用

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.

Guaranteed

Tested applications are guaranteed to work and covered by our Abpromise guarantee.

Predicted

Predicted to work for this combination of applications and species but not guaranteed.

Incompatible

Does not work for this combination of applications and species.

应用 Species
Flow Cyt
Human
ICC/IF
Human
IHC-P
Mouse
IP
Human
WB
Human
应用 Ab评论 说明
IHC-P (3)
1/100 - 1/200. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
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.

Flow Cyt
1/10 - 1/1000.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

IP
1/50.
说明
IHC-P
1/100 - 1/200. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
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.

Flow Cyt
1/10 - 1/1000.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

IP
1/50.

靶标

  • 功能

    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.
  • 组织特异性

    Ubiquitously expressed. Isoform 2 is also expressed in ovarian cancers.
  • 疾病相关

    Lung cancer
    Inflammatory skin and bowel disease, neonatal, 2
  • 序列相似性

    Belongs to the protein kinase superfamily. Tyr protein kinase family. EGF receptor subfamily.
    Contains 1 protein kinase domain.
  • 翻译后修饰

    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.
  • 细胞定位

    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
  • 数据库链接

    • 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
    • Unigene: 37227 Rat
    see all
  • 别名

    • 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

图片

  • 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

  • Western blot - Anti-EGFR antibody [E235] (ab32077)
    Western blot - Anti-EGFR antibody [E235] (ab32077)
    All lanes : Anti-EGFR antibody [E235] (ab32077)

    Lane 1 : A431 cell lysate
    Lane 2 : MDA-MB-468 cell lysate
    Lane 3 : Wild-type HeLa cell lysate
    Lane 4 : EGFR knockout HeLa cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 134 kDa



    Lanes 1 - 4: Merged signal (red and green). Green - ab32077 observed at 175 kDa. Red - loading control, ab130007 observed at 125 kDa.  

     ab32077 was shown to react with EGFR in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab255385 (knockout cell lysate ab263845) was used. Wild-type and EGFR knockout samples were subjected to SDS-PAGE. ab32077 and Anti-Vinculin antibody [VIN-54] (ab130007) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

     

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

实验方案

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

数据表及文件

  • SDS download

  • Datasheet download

    Download

文献 (10)

发表研究结果有使用 ab32077?请让我们知道,以便我们可以引用本数据表中的参考文章。

ab32077 被引用在 10 文献中.

  • Ayanlaja AA  et al. Doublecortin undergo nucleocytoplasmic transport via the RanGTPase signaling to promote glioma progression. Cell Commun Signal 18:24 (2020). PubMed: 32050972
  • Nagpal A  et al. Neoadjuvant neratinib promotes ferroptosis and inhibits brain metastasis in a novel syngeneic model of spontaneous HER2+ve breast cancer metastasis. Breast Cancer Res 21:94 (2019). PubMed: 31409375
  • Spence JS  et al. IFITM3 directly engages and shuttles incoming virus particles to lysosomes. Nat Chem Biol 15:259-268 (2019). PubMed: 30643282
  • Shao B  et al. The 3p14.2 tumour suppressor ADAMTS9 is inactivated by promoter CpG methylation and inhibits tumour cell growth in breast cancer. J Cell Mol Med 22:1257-1271 (2018). WB ; Human . PubMed: 29193730
  • Mansour AM  et al. Epidermal growth factor expression as a predictor of chemotherapeutic resistance in muscle-invasive bladder cancer. BMC Urol 18:100 (2018). PubMed: 30413194
  • Randall EC  et al. Integrated mapping of pharmacokinetics and pharmacodynamics in a patient-derived xenograft model of glioblastoma. Nat Commun 9:4904 (2018). PubMed: 30464169
  • Tian C  et al. ABCG1 as a potential oncogene in lung cancer. Exp Ther Med 13:3189-3194 (2017). PubMed: 28588672
  • Gao L  et al. Efficient Generation of Mice with Consistent Transgene Expression by FEEST. Sci Rep 5:16284 (2015). PubMed: 26573149
  • Xu X  et al. Establishment and characterization of GCSR1, a multi-drug resistant signet ring cell gastric cancer cell line. Int J Oncol 46:2479-87 (2015). PubMed: 25892440
  • Hofmann K  et al. A novel alkyne cholesterol to trace cellular cholesterol metabolism and localization. J Lipid Res 55:583-91 (2014). PubMed: 24334219

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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

Mr. Carl Hobbs

Verified customer

提交于 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

Mr. Carl Hobbs

Verified customer

提交于 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

Mr. Carl Hobbs

Verified customer

提交于 Jan 19 2018

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