Key Product Details

Validated by

Knockout/Knockdown

Species Reactivity

Validated:

Human

Cited:

Human, Primate

Applications

Validated:

Knockout Validated, Immunohistochemistry, Western Blot, Flow Cytometry, CyTOF-ready

Cited:

Immunohistochemistry, Immunohistochemistry-Paraffin, Immunohistochemistry-Frozen, Western Blot, Flow Cytometry, Immunocytochemistry, Functional Assay

Label

Unconjugated

Antibody Source

Polyclonal Goat IgG
View all EphA2 Primary Antibodies »

Product Specifications

Immunogen

Mouse myeloma cell line NS0-derived recombinant human EphA2
Gln25-Asn534
Accession # P29317

Specificity

Detects human EphA2 in direct ELISAs and Western blots. In direct ELISAs, less than 45% cross-reactivity with recombinant mouse EphA2 is observed and less than 1% cross-reactivity with recombinant human (rh) EphA1, rhEphA3, rhEphA4, rhEphA5, rhEphA6, rhEphA7 and rhEphA10 is observed.

Clonality

Polyclonal

Host

Goat

Isotype

IgG

Scientific Data Images for Human EphA2 Antibody

Detection of Human EphA2 antibody by Western Blot.

Detection of Human EphA2 by Western Blot.

Western blot shows lysates of A431 human epithelial carcinoma parental cell line and EphA2 knock out (KO) A431 cell line. PVDF membrane was probed with 1 µg/mL of Goat Anti-Human EphA2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3035) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (HAF017). A specific band was detected for EphA2 at approximately 110 kDa (as indicated), but not detectable in the knockout A431 cell line. GAPDH (Catalog # MAB5718) is shown as a loading control. This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of EphA2 antibody in A431 Human Cell Line antibody by Flow Cytometry.

Detection of EphA2 in A431 Human Cell Line by Flow Cytometry.

A431 human epithelial carcinoma cell line was stained with Goat Anti-Human EphA2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3035, filled histogram) or isotype control antibody (Catalog # AB-108-C, open histogram), followed by Phycoerythrin-conjugated Anti-Goat IgG Secondary Antibody (Catalog # F0107). View our protocol for Staining Membrane-associated Proteins.

EphA2 antibody in Human Ovarian Cancer Tissue by Immunohistochemistry (IHC-P).

EphA2 in Human Ovarian Cancer Tissue.

EphA2 was detected in immersion fixed paraffin-embedded sections of human ovarian cancer tissue using Goat Anti-Human EphA2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3035) at 5 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS008) and counterstained with hematoxylin (blue). Specific labeling was localized to the plasma membrane of cancer cells. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

Detection of Human EphA2 by Western Blot

Detection of Human EphA2 by Western Blot

Canonical EphA2-pY588 activation remains unaltered or is even increased after depletion of EFNA5. (A) Heat map for ephrin ligands and Eph receptor mRNAs in selected HGSC cell lines. Data obtained from CCLE and illustrated per cell line. (B, C) Quantification of EFNA1 (B) and EFNA5 (C) mRNAs in OVCAR3 and OVCAR4 with silenced EFNA1 or EFNA5 compared to the controls. N = 3. siScr is set to one. (D–G) EphA2 (total and phosphorylated) in OVCAR3 and OVCAR4 after silencing of EFNA1 or EFNA5 (D) along with EphA2-pY588 (E), EphA2-pS897 (F), and EphA2 (G) quantifications. N = 3. siScr is set to one. Full-length blots are presented in Supplementary Fig. 5. p values (Student’s t-test): *< 0.05; **< 0.01. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/33893375), licensed under a CC-BY license. Not internally tested by R&D Systems.

Applications for Human EphA2 Antibody

Application
Recommended Usage

CyTOF-ready

Ready to be labeled using established conjugation methods. No BSA or other carrier proteins that could interfere with conjugation.

Flow Cytometry

0.25 µg/106 cells
Sample: A431 human epithelial carcinoma cell line

Immunohistochemistry

5-15 µg/mL
Sample: Immersion fixed paraffin-embedded sections of human breast, ovarian, and pancreatic cancer tissue

Knockout Validated

1 µg/mL
Sample: A431 human epithelial carcinoma parental cell line and EphA2 knock out A431 cell line

Western Blot

1 µg/mL
Sample: A431 human epithelial carcinoma cell line

Reviewed Applications

Read 1 review rated 4 using AF3035 in the following applications:

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Flow Cytometry

Formulation, Preparation, and Storage

Purification

Antigen Affinity-purified

Reconstitution

Reconstitute at 0.2 mg/mL in sterile PBS. For liquid material, refer to CoA for concentration.

Formulation

Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.

Shipping

Lyophilized product is shipped at ambient temperature. Liquid small pack size (-SP) is shipped with polar packs. Upon receipt, store immediately at the temperature recommended below.

Stability & Storage

Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.

Calculators

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: EphA2

EphA2, also known as Eck, Myk2, and Sek2, is a member of the Eph receptor tyrosine kinase family which binds Ephrins A1, 2, 3, 4, and 5 (1-4). A and B class Eph proteins have a common structural organization. The human EphA2 cDNA encodes a 976 amino acid (aa) precursor including a 24 aa signal sequence, a 510 aa extracellular domain (ECD), a 24 aa transmembrane segment, and a 418 aa cytoplasmic domain. The ECD contains an N-terminal globular domain, a cysteine-rich domain, and two fibronectin type III domains (5). The cytoplasmic domain contains a juxtamembrane motif with two tyrosine residues, which are the major autophosphorylation sites, a kinase domain, and a sterile alpha motif (SAM) (5). The ECD of human EphA2 shares 90-94% aa sequence identity with mouse, bovine, and canine EphA2, and approximately 45% aa sequence identity with human EphA1, 3, 4, 5, 7, and 8. EphA2 becomes autophosphorylated following ligand binding (6, 7) and then interacts with SH2 domain-containing PI3-kinase to activate MAPK pathways (8, 9). Reverse signaling is also propagated through the Ephrin ligand. Transcription of EphA2 is dependent on the expression of E-Cadherin (10), and can be induced by p53 family transcription factors (11). EphA2 is upregulated in breast, prostate, and colon cancer vascular endothelium. Its ligand, EphrinA1, is expressed by the local tumor cells (12, 13). In some cases, EphA2 and EphrinA1 are expressed on the same blood vessels (14). EphA2 signaling cooperates with VEGF receptor signaling in promoting endothelial cell migration (13). The gene encoding human EphA2 maps to a region on chromosome 1 which is frequently deleted in neuroectodermal tumors (15).

References

  1. Poliakov, A. et al. (2004) Dev. Cell 7:465.
  2. Surawska, H. et al. (2004) Cytokine Growth Factor Rev. 15:419. 
  3. Pasquale, E.B. (2005) Nat. Rev. Mol. Cell Biol. 6:462.
  4. Davy, A. and P. Soriano (2005) Dev. Dyn. 232:1.
  5. Bohme, B et al. (1993) Oncogene 8:2857.
  6. Pandey, A. et al. (1995) Science 268:567.
  7. Bartley, T.D. et al. (1994) Nature 368:558.
  8. Pandey, A. et al. (1994) J. Biol. Chem. 269:30154.
  9. Miao, H. et al. (2001) Nat. Cell Biol. 3:527.
  10. Orsulic, S. and R. Kemler (2000) J. Cell Sci. 113:1793.
  11. Dohn, M. et al. (2001) Oncogene 20:6503.
  12. Zelinski, D.P. et al. (2001) Cancer Res. 61:2301.
  13. Brantley, D.M. et al. (2002) Oncogene 21:7011.
  14. Ogawa, K. et al. (2000) Oncogene 19:6043.
  15. Sulman, E.P. et al. (1997) Genomics 40:371.

Alternate Names

Eck, Myk2, Sek2

Entrez Gene IDs

1969 (Human); 13836 (Mouse)

Gene Symbol

EPHA2

UniProt

P29317

Additional EphA2 Products

Product Documents for Human EphA2 Antibody

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Product Specific Notices for Human EphA2 Antibody

For research use only

Citations for Human EphA2 Antibody

Customer Reviews for Human EphA2 Antibody (1)

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    Sample Tested: Serum and Plasma
    Species: Human
    Verified Customer | Posted 07/05/2022
    Human EphA2 Antibody AF3035

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