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Anti-EGFR antibody [EP38Y] is a rabbit recombinant monoclonal antibody that is used to detect EGFR in Flow cytometry (Intra), I-ELISA, ICC/IF, IHC-P, IP, Western blot. Suitable for Human, Mouse, Rat samples.

- Recognises total EGFR
- Specificity confirmed with EGFR knockout cell line validation
- Recombinant format for unrivaled batch-batch consistency
- Cited in over 340 publications


Images

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [EP38Y] (AB52894), expandable thumbnail
  • Immunoprecipitation - Anti-EGFR antibody [EP38Y] (AB52894), expandable thumbnail
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [EP38Y] (AB52894), expandable thumbnail
  • Western blot - Anti-EGFR antibody [EP38Y] (AB52894), expandable thumbnail
  • Western blot - Anti-EGFR antibody [EP38Y] (AB52894), expandable thumbnail

Publications

Key facts

Isotype
IgG
Host species
Rabbit
Storage buffer

pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA

Form
Liquid
Clonality
Monoclonal

Immunogen

  • The exact immunogen used to generate this antibody is proprietary information.

Reactivity data

Select an application
Product promiseTestedExpectedPredictedNot recommended
IPI-ELISAWBICC/IFFlow Cyt (Intra)IHC-P
Human
Tested
Expected
Tested
Tested
Tested
Tested
Mouse
Expected
Expected
Tested
Expected
Expected
Expected
Rat
Expected
Expected
Tested
Expected
Expected
Expected
Recombinant full length protein - Human
Not recommended
Tested
Not recommended
Not recommended
Not recommended
Not recommended

Tested
Tested

Species
Human
Dilution info
1/20
Notes

Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Expected
Expected

Species
Mouse, Rat
Dilution info
Use at an assay dependent concentration.
Notes

-

Not recommended
Not recommended

Species
Recombinant full length protein - Human
Dilution info
-
Notes

-

Tested
Tested

Species
Recombinant full length protein - Human
Dilution info
1/2500
Notes

-

Expected
Expected

Species
Human, Mouse, Rat
Dilution info
Use at an assay dependent concentration.
Notes

-

Tested
Tested

Species
Mouse
Dilution info
1/1000 - 1/10000
Notes

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

Species
Rat
Dilution info
1/1000 - 1/10000
Notes

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

Species
Human
Dilution info
1/1000 - 1/10000
Notes

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

Not recommended
Not recommended

Species
Recombinant full length protein - Human
Dilution info
-
Notes

-

Tested
Tested

Species
Human
Dilution info
1/250 - 1/500
Notes

-

Expected
Expected

Species
Mouse, Rat
Dilution info
Use at an assay dependent concentration.
Notes

-

Not recommended
Not recommended

Species
Recombinant full length protein - Human
Dilution info
-
Notes

-

Tested
Tested

Species
Human
Dilution info
1/20
Notes

Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

Expected
Expected

Species
Mouse, Rat
Dilution info
Use at an assay dependent concentration.
Notes

-

Not recommended
Not recommended

Species
Recombinant full length protein - Human
Dilution info
-
Notes

-

Tested
Tested

Species
Human
Dilution info
1/100
Notes

The mouse and rat recommendation is based on the WB results. This antibody may not be suitable for IHC with mouse or rat samples.

Perform heat-mediated antigen retrieval before commencing with IHC staining protocol.

Expected
Expected

Species
Mouse, Rat
Dilution info
Use at an assay dependent concentration.
Notes

-

Not recommended
Not recommended

Species
Recombinant full length protein - Human
Dilution info
-
Notes

-

Associated Products

Select an associated product type

10 products for Alternative Product

1 product for Alternative Version

Target data

Function

Receptor tyrosine kinase binding ligands of the EGF family and activating several signaling cascades to convert extracellular cues into appropriate cellular responses (PubMed:10805725, PubMed:27153536, PubMed:2790960, PubMed:35538033). Known ligands include EGF, TGFA/TGF-alpha, AREG, epigen/EPGN, BTC/betacellulin, epiregulin/EREG and HBEGF/heparin-binding EGF (PubMed:12297049, PubMed:15611079, PubMed:17909029, PubMed:20837704, PubMed:27153536, PubMed:2790960, PubMed:7679104, PubMed:8144591, PubMed:9419975). 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 (PubMed:27153536). May also activate the NF-kappa-B signaling cascade (PubMed:11116146). 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 (PubMed:11602604). Also phosphorylates MUC1 and increases its interaction with SRC and CTNNB1/beta-catenin (PubMed:11483589). Positively regulates cell migration via interaction with CCDC88A/GIV which retains EGFR at the cell membrane following ligand stimulation, promoting EGFR signaling which triggers cell migration (PubMed:20462955). Plays a role in enhancing learning and memory performance (By similarity). Plays a role in mammalian pain signaling (long-lasting hypersensitivity) (By similarity). Isoform 2 may act as an antagonist of EGF action. (Microbial infection) Acts as a receptor for hepatitis C virus (HCV) in hepatocytes and facilitates its cell entry. Mediates HCV entry by promoting the formation of the CD81-CLDN1 receptor complexes that are essential for HCV entry and by enhancing membrane fusion of cells expressing HCV envelope glycoproteins.

Alternative names

Recommended products

Anti-EGFR antibody [EP38Y] is a rabbit recombinant monoclonal antibody that is used to detect EGFR in Flow cytometry (Intra), I-ELISA, ICC/IF, IHC-P, IP, Western blot. Suitable for Human, Mouse, Rat samples.

- Recognises total EGFR
- Specificity confirmed with EGFR knockout cell line validation
- Recombinant format for unrivaled batch-batch consistency
- Cited in over 340 publications

Key facts

Isotype
IgG
Form
Liquid
Clonality
Monoclonal
Immunogen
  • The exact immunogen used to generate this antibody is proprietary information.
Clone number
EP38Y
Purification technique
Affinity purification Protein A
Specificity

The immunogen for this product is a synthetic phospho-peptide corresponding to residues surrounding Tyr1068 of human EGFR. After screening, clone EP38Y was found to recognize total EGFR and is not specific to phosphorylated-Tyr1068 EGFR. 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.
The mouse and rat recommendation is based on the WB results. This antibody may not be suitable for IHC with mouse or rat samples.

Dissociation constant
1.9 x 10-11 M
Concentration
Loading...

Storage

Shipped at conditions
Blue Ice
Appropriate short-term storage duration
1-2 weeks
Appropriate short-term storage conditions
+4°C
Appropriate long-term storage conditions
-20°C
Aliquoting information
Upon delivery aliquot
Storage information
Avoid freeze / thaw cycle

Notes

Anti-EGFR antibody [EP38Y] (ab52894) was developed by Abcam using patented rabbit monoclonal antibody technology and is validated for use in Flow Cyt (Intra), I-ELISA, ICC/IF, IHC-P, IP and WB.

Anti-EGFR antibody [EP38Y] (ab52894) was first used in a scientific publication in 2010 and has been cited over 342 times in peer reviewed journals. It's performance in Western Blot and IHC in human samples is trusted by the scientific community.

Abcam's high quality manufacturing and validation processes ensure Anti-EGFR antibody [EP38Y] (ab52894) has high sensitivity and specificity alongside high lot-to-lot consistency and reproducibility.

The specificity of Anti-EGFR antibody [EP38Y] (ab52894) has been confirmed by Western Blot testing in EGFR knockout HeLa cells (Human EGFR knockout HeLa cell line ab255385).

Anti-EGFR antibody [EP38Y] (ab52894) has 22 independent reviews from customers.

Anti-EGFR antibody [EP38Y] (ab52894) specifically detects EGFR (UniProt ID: P00533; Molecular weight: 133kDa) and is sold in a convenient trial size to enable initial testing (20 µL) and larger sizes for subsequent scaling up experiments (100 µL and 1 mL).

Conjugation-ready, carrier free format available for antibody clone EP38Y - Anti-EGFR antibody [EP38Y] - Low endotoxin, Azide free ab174481.

Antibody clone EP38Y is also available pre-conjugated to a variety of labels for your convenience - Alexa Fluor® 647, Alexa Fluor® 488, HRP, Alexa Fluor® 594, PE, Alexa Fluor® 555, Biotin, Alexa Fluor® 750 (Alexa Fluor® 647 Anti-EGFR antibody [EP38Y] ab192982, Alexa Fluor® 488 Anti-EGFR antibody [EP38Y] ab193244, HRP Anti-EGFR antibody [EP38Y] ab193602, Alexa Fluor® 594 Anti-EGFR antibody [EP38Y] ab207870, PE Anti-EGFR antibody [EP38Y] ab208753, Alexa Fluor® 555 Anti-EGFR antibody [EP38Y] ab274892, Biotin Anti-EGFR antibody [EP38Y] ab314262, Alexa Fluor® 750 Anti-EGFR antibody [EP38Y] ab321669).

The epidermal growth factor receptor (EGFR) is a critical player in oncology due to its role in cell proliferation, survival and differentiation. It is a receptor tyrosine kinase that, when mutated or overexpressed, can drive the development and progression of various cancers, including non-small cell lung cancer (NSCLC) and glioblastoma.

Patented technology
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.
Activity summary

EGFR or Epidermal Growth Factor Receptor is a transmembrane glycoprotein that acts as a receptor for members of the epidermal growth factor family. Known alternatively as ErbB1 or HER1 this receptor has an approximate molecular weight of 170 kDa. EGFR is expressed in various cell types notably on epithelial cells and can influence multiple cellular processes through its kinase activity. It participates in the regulation of cell growth multiplication and survival by activating its kinase domain upon ligand binding.

Biological function summary

The EGFR protein plays an important role in cellular communication and signaling processes. EGFR pairs with other receptor family members to form active dimers or even higher-order complexes which in turn initiate intracellular signaling cascades. Through these complexes EGFR influences many processes including cell differentiation and repair. This function of EGFR makes it an integral part of mammalian biology affecting how cells respond to their environment by mediating changes in gene expression.

Pathways

EGFR is a central player in the MAPK and PI3K/Akt signaling pathways. Alongside other protein partners like KRAS and PI3 kinase it contributes to transmitting signals from the cell surface to the nucleus affecting gene transcription and cell behavior. These pathways are important for normal cell growth and division and aberrations in these pathways can lead to excessive or insufficient cell proliferation.

Associated diseases and disorders

EGFR is pertinent to cancer biology including non-small cell lung cancer and glioblastoma where mutations or overexpression of the receptor frequently occur. It connects to proteins such as PTEN and BRAF which influence tumor progression and response to targeted therapies. EGFR's involvement in these disorders highlights its significance as a therapeutic target since it can be manipulated to alter disease progression.

Product promise

We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.

In the unlikely event of one of our products not working as expected, you are covered by our product promise.

Full details and terms and conditions can be found here:
Terms & Conditions.

21 product images

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [EP38Y] (ab52894), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [EP38Y] (ab52894)

    EGFR Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) staining of human placenta using rabbit Anti-EGFR antibody

    Immunohistochemical analysis of formalin fixed paraffin embedded human placenta labelling EGFR with ab52894 at a concentration of 0.21 µg/ml. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with a Bond™ Polymer Refine Detection kit. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution 2) for 20 mins. ab52894 Anti-EGFR antibody [EP38Y] was incubated for 30 mins at room temperature. Sections were counterstained with Hematoxylin. Image inset shows absence of staining in secondary antibody only control.

  • Immunoprecipitation - Anti-EGFR antibody [EP38Y] (ab52894), expandable thumbnail

    Immunoprecipitation - Anti-EGFR antibody [EP38Y] (ab52894)

    ab52894 (purified) at 1:20 dilution (0.5 μg) immunoprecipitating EGFR in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate.

    Lane 1 (input): HeLa whole cell lysate 10 μg

    Lane 2 (+): ab52894 in HeLa whole cell lysate

    Lane 3 (-): Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab52894 in HeLa whole cell lysate

    For western blotting, VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) was used for detection at 1:1000 dilution.

    Blocking and diluting buffer: 5% NFDM/TBST.

    All lanes: Immunoprecipitation - Anti-EGFR antibody [EP38Y] (ab52894)

    Predicted band size: 134 kDa

    Observed band size: 175 kDa

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [EP38Y] (ab52894), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [EP38Y] (ab52894)

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue sections labeling EGFR with purified ab52894 at 1:100 dilution (0.95 μg/ml).

    Heat mediated antigen retrieval was performed using EDTA buffer, pH 9.0. Tissue was counterstained with hematoxylin. Goat Anti-Rabbit IgG H&L (HRP) ab97051 Goat Anti-Rabbit IgG H&L (HRP) secondary antibody was used at 1:500 dilution. PBS instead of the primary antibody was used as the negative control.

  • Western blot - Anti-EGFR antibody [EP38Y] (ab52894), expandable thumbnail

    Western blot - Anti-EGFR antibody [EP38Y] (ab52894)

    EGFR Western blot staining using rabbit Anti-EGFR antibody

    False colour image of Western blot: Anti-EGFR antibody [EP38Y] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab52894 was shown to bind specifically to EGFR. A band was observed at 160 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in Egfr CRISPR-Cas9 edited cell line Human EGFR knockout HCT116 cell line ab281597 (CRISPR-Cas9 edited cell lysate ab282949). The band observed in the CRISPR-Cas9 edited lysate lane below 160 kDa is likely to represent a truncated form of EGFR. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and Egfr CRISPR-Cas9 edited 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 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.

    All lanes: Western blot - Anti-EGFR antibody [EP38Y] (ab52894) at 1/1000 dilution

    Lane 1: Wild-type HCT 116 cell lysate at 20 µg

    Lane 2: EGFR CRISPR-Cas9 edited HCT 116 cell lysate at 20 µg

    Lane 3: Caco-2 cell lysate at 20 µg

    Performed under reducing conditions.

    Predicted band size: 134 kDa

    Observed band size: 160 kDa

  • Western blot - Anti-EGFR antibody [EP38Y] (ab52894), expandable thumbnail

    Western blot - Anti-EGFR antibody [EP38Y] (ab52894)

    EGFR Western blot staining using rabbit Anti-EGFR antibody

    > Lanes 1 - 4: Merged signal (red and green). Green - ab52894 observed at 175 kDa. Red - loading control, Anti-Vinculin antibody [VIN-54] ab130007 observed at 125 kDa.

    ab52894 was shown to react with EGFR in wild-type HeLa. Loss of signal was observed when knockout cell line Human EGFR knockout HeLa cell line ab255385 (knockout cell lysate Human EGFR knockout HeLa cell lysate ab263845) was used. Wild-type and EGFR knockout samples were subjected to SDS-PAGE. ab52894 and Anti-Vinculin antibody [VIN-54] (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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (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.

    All lanes: Western blot - Anti-EGFR antibody [EP38Y] (ab52894) at 1/1000 dilution

    Lane 1: A431 cell lysate at 20 µg

    Lane 2: MDA-MB-468 cell lysate at 20 µg

    Lane 3: Wild-type HeLa cell lysate at 20 µg

    Lane 4: EGFR knockout HeLa cell lysate at 20 µg

    Secondary

    All lanes: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution

    Performed under reducing conditions.

    Predicted band size: 134 kDa

    Observed band size: 124 kDa, 134 kDa

  • Western blot - Anti-EGFR antibody [EP38Y] (ab52894), expandable thumbnail

    Western blot - Anti-EGFR antibody [EP38Y] (ab52894)

    EGFR Western blot staining using rabbit Anti-EGFR antibody

    Blocking and diluting buffer: 5% NFDM/TBST

    All lanes: Western blot - Anti-EGFR antibody [EP38Y] (ab52894) at 1/10000 dilution

    Lane 1: Rat liver lysates at 15 µg

    Lane 2: Mouse lung lysates at 15 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

    Predicted band size: 134 kDa

    Observed band size: 175 kDa

  • Flow Cytometry (Intracellular) - Anti-EGFR antibody [EP38Y] (ab52894), expandable thumbnail

    Flow Cytometry (Intracellular) - Anti-EGFR antibody [EP38Y] (ab52894)

    Intracellular Flow Cytometry analysis of A431 (Human epidermoid carcinoma epithelial cell) cells labeling EGFR with purified ab52894.

    Cells were fixed with 4% paraformaldehyde (10 mins) and permeabilized with 90% methanol for 30 mins. Then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by ab52894 at 1/20 dilution (red) for 30 mins. A Goat anti rabbit IgG (Alexa Fluorr® 488) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

  • Western blot - Anti-EGFR antibody [EP38Y] (ab52894), expandable thumbnail

    Western blot - Anti-EGFR antibody [EP38Y] (ab52894)

    EGFR Western blot staining of HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates using rabbit Anti-EGFR antibody

    Blocking and diluting buffer: 5% NFDM/TBST

    All lanes: Western blot - Anti-EGFR antibody [EP38Y] (ab52894) at 1/2000 dilution

    All lanes: HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 20 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

    Predicted band size: 134 kDa

    Observed band size: 175 kDa

  • Immunocytochemistry/ Immunofluorescence - Anti-EGFR antibody [EP38Y] (ab52894), expandable thumbnail

    Immunocytochemistry/ Immunofluorescence - Anti-EGFR antibody [EP38Y] (ab52894)

    Immunocytochemistry/ Immunofluorescence analysis of A431 (Human epidermoid carcinoma epithelial cell) cells labeling EGFR with purified ab52894 at 1:250 dilution (0.4 μg/ml).

    Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5 μg/ml). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat anti rabbit IgG(Alexa Fluor® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain.

    PBS instead of the primary antibody was used as the secondary antibody only control.

  • Indirect ELISA - Anti-EGFR antibody [EP38Y] (ab52894), expandable thumbnail

    Indirect ELISA - Anti-EGFR antibody [EP38Y] (ab52894)

    ELISA analysis of Human EGFR recombinant protein at 1000 ng/ml with ab52894. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2500 dilution was used as the secondary antibody.

  • Western blot - Anti-EGFR antibody [EP38Y] (ab52894), expandable thumbnail

    Western blot - Anti-EGFR antibody [EP38Y] (ab52894)

    EGFR Western blot staining using rabbit Anti-EGFR antibody

    This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 mins before being transferred onto a Nitrocellulose membrane at 30V for 70 mins. The membrane was then blocked for an hour before being incubated with unpurified ab52894 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-labeled 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.

    All lanes: Western blot - Anti-EGFR antibody [EP38Y] (ab52894) at 1/1000 dilution

    Lane 1: Caco-2 (Human colorectal adenocarcinoma cell line) cell lysate at 20 µg

    Lane 2: A431 (Human epidermoid carcinoma cell line) cell lysate at 20 µg

    Lane 3: Mouse skin cell lysate at 20 µg

    Lane 4: Rat skin cell lysate at 20 µg

    Performed under reducing conditions.

    Predicted band size: 134 kDa

  • OI-RD Scanning - Anti-EGFR antibody [EP38Y] (ab52894), expandable thumbnail

    OI-RD Scanning - Anti-EGFR antibody [EP38Y] (ab52894)

    We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody.
    Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.

  • Western blot - Anti-EGFR antibody [EP38Y] (ab52894), expandable thumbnail

    Western blot - Anti-EGFR antibody [EP38Y] (ab52894)

    EGFR Western blot staining using rabbit Anti-EGFR antibody

    Different batches of ab52894 were tested on HeLa (Human cervix adenocarcinoma epithelial cell) lysate at 1.0 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 175 kDa.

    All lanes: Western blot - Anti-EGFR antibody [EP38Y] (ab52894)

    Predicted band size: 134 kDa

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [EP38Y] (ab52894), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [EP38Y] (ab52894)

    EGFR Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) staining using rabbit Anti-EGFR antibody

    Tissue microarrays stained for Anti-EGFR antibody [EP38Y] using ab52894 in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negative (cross mark) staining per sample type tested. The sections were pre-treated using heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins. The section was incubated with ab52894 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument

  • Immunocytochemistry/ Immunofluorescence - Anti-EGFR antibody [EP38Y] (ab52894), expandable thumbnail

    Immunocytochemistry/ Immunofluorescence - Anti-EGFR antibody [EP38Y] (ab52894)

    EGFR Immunocytochemistry/ Immunofluorescence staining using rabbit Anti-EGFR antibody

    ab52894 staining of EGFR in a HCT116 cell spheroid. The cells were fixed with 4% formaldehyde (10 min), permeabilised with 0.5% Triton X-100 for 1h and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween overnight at room temperature. The spheroids were then incubated overnight at room temperature with ab52894 at 2 μg/ml and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, Mouse monoclonal [DM1A] to alpha Tubulin at 2 μg/ml. DAPI was used as nuclear counterstain (shown in blue). As secondary antibodies Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat anti-Rabbit (Alexa Fluor® 488) (shown in green) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 Goat anti-Mouse (Alexa Fluor® 594) (shown in magenta) were used, incubated overnight at room temperature. All permeabilization, blocking and antibody incubation steps were performed using a rotary shaker.

    Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

    The antibody ab52894 also worked using 100% methanol (5 min).

  • Western blot - Anti-EGFR antibody [EP38Y] (ab52894), expandable thumbnail

    Western blot - Anti-EGFR antibody [EP38Y] (ab52894)

    EGFR Western blot staining using rabbit Anti-EGFR antibody

    Western blot: Anti-EGFR antibody [EP38Y] (ab52894) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab52894 was shown to bind specifically to EGFR. A band was observed at 175 kDa in wild-type A549 cell lysates with no signal observed at this size in EGFR knockout cell line. To generate this image, wild-type and EGFR knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5% milk in TBS-0.1% Tween® 20 (TBS-T) 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

    All lanes: Western blot - Anti-EGFR antibody [EP38Y] - BSA and Azide free (Anti-EGFR antibody [EP38Y] - BSA and Azide free ab272293)

    Lane 1: Wild-type A549 cell lysate at 20 µg

    Lane 2: EGFR knockout A549 cell lysate at 20 µg

    Lane 3: Wild-type HeLa cell lysate at 20 µg

    Lane 4: EGFR knockout HeLa cell lysate at 20 µg

    Developed using the ECL technique.

    Performed under reducing conditions.

    Observed band size: 175 kDa

  • Western blot - Anti-EGFR antibody [EP38Y] (ab52894), expandable thumbnail

    Western blot - Anti-EGFR antibody [EP38Y] (ab52894)

    EGFR Western blot staining using rabbit Anti-EGFR antibody

    Blocking and diluting buffer and concentration: 5% NFDM/TBST.

    In Western blot, Anti-EGFR (phospho Y845 + Y1068 + Y1086) antibody [RM1132] ab319113 was shown to bind specifically to EGFR. Target of interest was observed at 175kDa, in wild-type HeLa cell lysates (lane 1/2/5/6) with no signal observed at this size in EGFR knockout cell line (lane 3/4/7/8) (knockout cell line Human EGFR knockout HeLa cell line ab255385 / knockout cell lysate Human EGFR knockout HeLa cell lysate ab263845).

    In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) staining at 1/200000 dilution.

    In Western blot, Anti-EGFR antibody [EP38Y] (ab52894) staining at 1/1000 dilution.

    All lanes: Western blot - Anti-EGFR (phospho Y845 + Y1068 + Y1086) antibody [RM1132] (Anti-EGFR (phospho Y845 + Y1068 + Y1086) antibody [RM1132] ab319113) at 1/1000 dilution

    Lane 1: Untreated wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate (untreated membrane) at 20 µg

    Lane 2: Wild-type HeLa treated with 100ng/ml EGF for 30 minutes whole cell lysate (untreated membrane) at 20 µg

    Lane 3: Untreated EGFR knockout HeLa whole cell lysate (untreated membrane) at 20 µg

    Lane 4: EGFR knockout HeLa treated with 100ng/ml EGF for 30 minutes whole cell lysate (untreated membrane) at 20 µg

    Lane 5: Untreated wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

    Lane 6: Wild-type HeLa treated with 100ng/ml EGF for 30 minutes whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

    Lane 7: Untreated EGFR knockout HeLa whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

    Lane 8: EGFR knockout HeLa treated with 100ng/ml EGF for 30 minutes whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

    Performed under reducing conditions.

    Observed band size: 175 kDa, 36 kDa

    Exposure time: 180s

  • Western blot - Anti-EGFR antibody [EP38Y] (ab52894), expandable thumbnail

    Western blot - Anti-EGFR antibody [EP38Y] (ab52894)

    EGFR Western blot staining using rabbit Anti-EGFR antibody

    Blocking and diluting buffer and concentration: 5% NFDM/TBST.

    In Western blot, Anti-Vinculin antibody [EPR8185] (Anti-Vinculin antibody [EPR8185] - Loading Control ab129002) staining at 1/10000 dilution.

    In Western blot, Anti-EGFR antibody [EP38Y] (ab52894) staining at 1/1000 dilution.

    All lanes: Western blot - Anti-EGFR (phospho Y845 + Y1068 + Y1086) antibody [RM1132] (Anti-EGFR (phospho Y845 + Y1068 + Y1086) antibody [RM1132] ab319113) at 1/5000 dilution

    Lane 1: Untreated A431 (human epidermoid carcinoma epithelial cell) whole cell lysate (untreated membrane) at 10 µg

    Lane 2: A431 treated with 100ng/ml EGF for 30 minutes whole cell lysate (untreated membrane) at 10 µg

    Lane 3: Untreated A431 whole cell lysate (alkaline phosphatase treated membrane) at 10 µg

    Lane 4: A431 treated with 100ng/ml EGF for 30 minutes whole cell lysate (alkaline phosphatase treated membrane) at 10 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

    Observed band size: 175 kDa, 124 kDa

    Exposure time: 169s

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [EP38Y] (ab52894), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [EP38Y] (ab52894)

    EGFR Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) staining of Human glioblastoma tissue using rabbit Anti-EGFR antibody

    Chromogenic duplex (2-plex) immunohistochemical staining of formalin fixed paraffin-embedded human glioblastoma tissue performed on a Roche Ventana Discovery Ultra instrument.

    Heat mediated antigen retrieval was performed with CC1 solution for 64 min at 95°C. Two rounds of primary antibody incubation were performed each for 16 min at 37°C. anti-EGFRvIII (Anti-EGFRvIII antibody [EPR28380-83] ab313646; 1st primary antibody; 3 µg/ml) and anti-EGFR (ab52894; 2nd primary antibody; 0.5 µg/ml). Antibody denaturation was conducted after the first round of staining using Ultra CC2 solution for 8 min at 100°C to avoid cross reactivity.

    Signal was developed with anti-rabbit HQ followed by anti-HQ HRP coupled with purple (Discovery purple kit (RUO) #760-229) and teal (Discovery teal kit (RUO) #760-247) chromogens and counterstained with haematoxylin II.

    Tissue obtained from the Human Research Tissue Bank, supported by the NHR Cambridge Biomedical Research Centre.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [EP38Y] (ab52894), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [EP38Y] (ab52894)

    EGFR Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) staining of human placenta using rabbit Anti-EGFR antibody

    Immunohistochemical analysis of formalin fixed paraffin embedded human placenta labelling EGFR with ab52894 at a concentration of 0.1 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 32 mins at 100°C with ULTRA cell conditioning solution (CC1, pH 8.5). ab52894 Anti-EGFR antibody [EP38Y] was incubated at 37°C for 16 mins. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [EP38Y] (ab52894), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EGFR antibody [EP38Y] (ab52894)

    EGFR Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) staining of Human glioblastoma and placenta tissues using rabbit Anti-EGFR antibody

    Immunohistochemical analysis of formalin fixed paraffin-embedded human glioblastoma and placenta tissues labelling EGFRvIII with Anti-EGFRvIII antibody [EPR28380-83] ab313646 and EGFR with ab52894 at a concentration of 2.5 µg/ml and 0.5 µg/ml.

    The immunostaining was performed on a Leica Biosystems BOND® RX instrument with a Bond™ Polymer Refine Detection kit. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution 2) for 20 mins. Primary antibodies were incubated for 30 mins at room temperature. Sections were counterstained with Hematoxylin.

    Tissue obtained from the Human Research Tissue Bank, supported by the NHR Cambridge Biomedical Research Centre.

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