EGFR Antibody (PD00-75)

Novus Biologicals | Catalog # NBP3-32255

Recombinant Monoclonal Antibody
Novus Biologicals
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Key Product Details

Species Reactivity

Human

Applications

Immunohistochemistry, Immunohistochemistry-Paraffin, Western Blot, Flow Cytometry, Immunocytochemistry/ Immunofluorescence

Label

Unconjugated

Antibody Source

Recombinant Monoclonal Rabbit IgG Clone # PD00-75 expressed in HEK293
View all EGFR Primary Antibodies »

Product Specifications

Immunogen

Synthetic peptide corresponding to C-terminus of human EGFR protein. (Uniprot: P00533)

Localization

Cell membrane, Endoplasmic reticulum membrane, Golgi apparatus membrane, Nucleus membrane, Endosome, Nucleus, Secreted.

Clonality

Monoclonal

Host

Rabbit

Isotype

IgG

Theoretical MW

134 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Scientific Data Images for EGFR Antibody (PD00-75)

EGFR Antibody (PD00-75)

Immunohistochemistry: EGFR Antibody (PD00-75) [NBP3-32255]

Immunohistochemistry: EGFR Antibody (PD00-75) [NBP3-32255] - Immunohistochemical analysis of paraffin-embedded human skin tissue with Rabbit anti-EGFR antibody (NBP3-32255) at 1/800 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (NBP3-32255) at 1/800 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
EGFR Antibody (PD00-75)

Immunocytochemistry/ Immunofluorescence: EGFR Antibody (PD00-75) [NBP3-32255]

Immunocytochemistry/ Immunofluorescence: EGFR Antibody (PD00-75) [NBP3-32255] - Immunocytochemistry analysis of A431 cells labeling EGFR with Rabbit anti-EGFR antibody (NBP3-32255) at 1/500 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-EGFR antibody at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594) was used as the secondary antibody at 1/1,000 dilution.
EGFR Antibody (PD00-75)

Flow Cytometry: EGFR Antibody (PD00-75) [NBP3-32255]

Flow Cytometry: EGFR Antibody (PD00-75) [NBP3-32255] - Flow cytometric analysis of A431 cells labeling EGFR.

Cells were fixed and permeabilized. Then stained with the primary antibody (NBP3-32255, red) at 1/1,000 dilution, compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with an iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody at 1/1,000 dilution for 30 minutes at +4℃. Unlabeled sample was used as a control (cells without incubation with primary antibody; black).
EGFR Antibody (PD00-75)

Western Blot: EGFR Antibody (PD00-75) [NBP3-32255]

Western Blot: EGFR Antibody (PD00-75) [NBP3-32255] - Western blot analysis of EGFR on different lysates with Rabbit anti-EGFR antibody (NBP3-32255) at 1/2,000 dilution.
Lane 1: HeLa cell lysate
Lane 2: A431 cell lysate
Lane 3: MDA-MB-468 cell lysate
Lane 4: MCF7 cell lysate (low expression)

Lysates/proteins at 15 µg/Lane.

Predicted band size: 134 kDa
Observed band size: 150 kDa

Exposure time: 21 seconds;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/50,000 dilution was used for 1 hour at room temperature.

Applications for EGFR Antibody (PD00-75)

Application
Recommended Usage

Flow Cytometry

1:1000

Immunocytochemistry/ Immunofluorescence

1:500

Immunohistochemistry-Paraffin

1:800

Western Blot

1:2000

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Flow Cytometry

Formulation, Preparation, and Storage

Purification

Protein A purified

Formulation

PBS (pH7.4), 0.1% BSA and 40% Glycerol

Preservative

0.05% Sodium Azide

Concentration

1 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.

Background: EGFR

Epidermal growth factor receptor (EGFR), also known as ErbB1 and HER1, is a type I glycoprotein that belongs the ErbB subfamily of receptor tyrosine kinases (RTKs), which includes ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4 (1,2). EGFR plays an important role in epithelial cell development and homeostasis and as a driver of tumorigenesis in cancer (1,2). The human EGFR is protein 1210 amino acids (aa) in length with a theoretical molecular weight (MW) of 134 kDa (1). The protein consists of a short signal peptide, an extracellular domain (ECD) divided into four subdomains (I-IV), a transmembrane region, an intracellular juxtamembrane segment, a tyrosine kinase domain, and C-terminal tail (1-3). Within the ECD, human EGFR has 88-90% aa sequence identity with mouse and rat EGFR. EGFR has four known specific ligands: EGF, amphiregulin, epigen, and transforming growth factor alpha (TGF-alpha). EGFR ligands betacellulin, epiregulin, and herapin binding (HB)-EGF have dual specificity with ErbB4 (1,3). Ligand binding to the extracellular domain of EFGR leads to receptor homodimerization, or heterodimerization with other ErbB family members, and EGFR activation. This results in subsequent phosphorylation and activation of intracellular signaling pathways, such as MAPK and PI3K/Akt (2,3). EGFR signaling is essential for many cellular processes including proliferation, differentiation, migration, and apoptosis (1,3,5).

In addition to its role in normal development, EGFR mutations or overexpression is observed in many tumors, including breast cancer, non-small cell lung carcinoma (NSCLC), colon cancer, and more (3-6). Small molecule tyrosine kinase inhibitors (TKIs), like gefitinib, erlotinib, and afatinib, have shown great efficacy in treating patients with EGFR activating mutations, especially for NSCLC (4-6). However, most patients eventually develop acquired resistance to TKIs and thus combination and alternative therapies are in development (4-6). A third-generation TKI, osimertinib, is approved for NSCLC patients with resistance to first-line EGFR TKI treatment (6). Additionally, combination therapies of EGFR TKIs with monoclonal antibody immunotherapies, like anti-PD-L1, are being further investigated in clinical trials (6).

References

1. Roskoski R Jr. Small molecule inhibitors targeting the EGFR/ErbB family of protein-tyrosine kinases in human cancers. Pharmacol Res. 2019; 139:395-411. https://doi.org/10.1016/j.phrs.2018.11.014

2. Sigismund S, Avanzato D, Lanzetti L. Emerging functions of the EGFR in cancer. Mol Oncol. 2018; 12(1):3-20. https://doi.org/10.1002/1878-0261.12155

3. Normanno N, De Luca A, Bianco C, et al. Epidermal growth factor receptor (EGFR) signaling in cancer. Gene. 2006; 366(1):2-16. https://doi.org/10.1016/j.gene.2005.10.018

4. Liu Q, Yu S, Zhao W, Qin S, Chu Q, Wu K. EGFR-TKIs resistance via EGFR-independent signaling pathways. Mol Cancer. 2018; 17(1):53. https://doi.org/10.1186/s12943-018-0793-1

5. Harrison PT, Vyse S, Huang PH. Rare epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer. Semin Cancer Biol. 2020; 61:167-179. https://doi.org/10.1016/j.semcancer.2019.09.015

6. Wu SG, Shih JY. Management of acquired resistance to EGFR TKI-targeted therapy in advanced non-small cell lung cancer. Mol Cancer. 2018; 17(1):38. https://doi.org/10.1186/s12943-018-0777-1

Long Name

Epidermal Growth Factor Receptor

Alternate Names

EGF R, ErbB, ErbB1, HER-1

Gene Symbol

EGFR

Additional EGFR Products

Product Documents for EGFR Antibody (PD00-75)

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Product Specific Notices for EGFR Antibody (PD00-75)

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

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Protocols

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