Key Product Details

Validated by

Knockout/Knockdown

Species Reactivity

Validated:

Human

Cited:

Human, Mouse

Applications

Validated:

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

Cited:

Immunohistochemistry, Immunohistochemistry-Frozen, Western Blot, Immunocytochemistry, Immunoprecipitation

Label

Unconjugated

Antibody Source

Polyclonal Goat IgG
View all EphB4 Primary Antibodies »

Product Specifications

Immunogen

Mouse myeloma cell line NS0-derived recombinant human EphB4
Leu16-Ala539
Accession # P54760

Specificity

Detects human EphB4 in direct ELISAs and Western blots. In direct ELISAs, less than 10% cross-reactivity with recombinant mouse (rm) EphB4 is observed and less than 1% cross-reactivity with recombinant human (rh) EphA4, rhEphA3, rhEphB2, and rhEphB3 is observed.

Clonality

Polyclonal

Host

Goat

Isotype

IgG

Scientific Data Images for Human EphB4 Antibody

Detection of Human EphB4 antibody by Western Blot.

Detection of Human EphB4 by Western Blot.

Western blot shows lysates of K562 human chronic myelogenous leukemia cell line, COLO 205 human colorectal adenocarcinoma cell line, ZR-75 human breast cancer cell line, and HUVEC human umbilical vein endothelial cells. PVDF membrane was probed with 2 µg/mL of Goat Anti-Human EphB4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3038) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). A specific band was detected for EphB4 at approximately 120 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of EphB4 antibody in MCF-7 Human Cell Line antibody by Flow Cytometry.

Detection of EphB4 in MCF‑7 Human Cell Line by Flow Cytometry.

MCF-7 human breast cancer cell line was stained with Goat Anti-Human EphB4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3038, 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.

EphB4 antibody in Human Kidney by Immunohistochemistry (IHC-P).

EphB4 in Human Kidney.

EphB4 was detected in immersion fixed paraffin-embedded sections of human kidney using 15 µg/mL Goat Anti-Human EphB4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3038) overnight at 4 °C. Tissue was stained with the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS008) and counterstained with hematoxylin (blue). View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

Detection of Human EphB4 antibody by Simple WesternTM.

Detection of Human EphB4 by Simple WesternTM.

Simple Western lane view shows lysates of HUVEC human umbilical vein endothelial cells, loaded at 0.2 mg/mL. A specific band was detected for EphB4 at approximately 127 kDa (as indicated) using 50 µg/mL of Goat Anti-Human EphB4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3038) followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Western Blot Shows Human EphB4 Antibody Specificity by Using Knockout Cell Line.

Western Blot Shows Human EphB4 Specificity by Using Knockout Cell Line.

Western blot shows lysates of HEK293T human embryonic kidney parental cell line and EphB4 knockout HEK293T cell line (KO). PVDF membrane was probed with 2 µg/mL of Goat Anti-Human EphB4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3038) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). A specific band was detected for EphB4 at approximately 140 kDa (as indicated) in the parental HEK293T cell line, but is not detectable in knockout HEK293T cell line. GAPDH (Catalog # AF5718) is shown as a loading control. This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Mouse EphB4 by Western Blot

Detection of Mouse EphB4 by Western Blot

EphB4 expression in transfected A375 melanoma cells and corresponding tumor xenografts. Western blot analysis of EphB4 and its ligand EphrinB2 in A375, A375-pIRES, and A375-EphB4 whole cell lysates (A) and tumor lysates (E). Anti-beta -actin served as loading control. (B) Relative mRNA expression of EphB4 and EphrinB2 in A375, A375-pIRES, and A375-EphB4 cells, analyzed by quantitative real-time RT-PCR was normalized to the constitutive expression level of beta -actin and to expression level in wild-type A375 melanoma cells using the delta delta CT method (2−∆∆ct) resulting in a value of 1 for A375 cells. Values represent mean ± SEM of at least three independent experiments each performed in triplicate (* p < 0.05, *** p < 0.001). (C) EphB4 phosphorylation was analyzed by pEphB4-ELISA in whole cell lysates of A375 EphB4 cells incubated with different concentrations of sEphrinB2-Fc (0; 0,5; 1 µg/mL) for 15, 30, and 60 min. Values represent mean ± SD from one of at least three independent experiments, each performed in duplicate. To rule out influence of sEphrinB2 on total EphB4 protein amount, EphB4 was analyzed in the same cell lysates by western blot analysis (figure shows one representative blot out of three independent experiments performed in duplicate) ranging from 92% to 112% of control as calculated after densitometric analysis. Anti beta -actin served as loading control. (D) Immunohistochemical detection of EphB4 in acetone-fixed cryosections of A375 pIRES and A375 EphB4 tumor xenografts using goat anti EphB4 antibody. Sections stained without the primary antibody served as negative control. Scale bar 50 µm. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/29462967), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse EphB4 by Western Blot

Detection of Mouse EphB4 by Western Blot

EphB4 expression in transfected A375 melanoma cells and corresponding tumor xenografts. Western blot analysis of EphB4 and its ligand EphrinB2 in A375, A375-pIRES, and A375-EphB4 whole cell lysates (A) and tumor lysates (E). Anti-beta -actin served as loading control. (B) Relative mRNA expression of EphB4 and EphrinB2 in A375, A375-pIRES, and A375-EphB4 cells, analyzed by quantitative real-time RT-PCR was normalized to the constitutive expression level of beta -actin and to expression level in wild-type A375 melanoma cells using the delta delta CT method (2−∆∆ct) resulting in a value of 1 for A375 cells. Values represent mean ± SEM of at least three independent experiments each performed in triplicate (* p < 0.05, *** p < 0.001). (C) EphB4 phosphorylation was analyzed by pEphB4-ELISA in whole cell lysates of A375 EphB4 cells incubated with different concentrations of sEphrinB2-Fc (0; 0,5; 1 µg/mL) for 15, 30, and 60 min. Values represent mean ± SD from one of at least three independent experiments, each performed in duplicate. To rule out influence of sEphrinB2 on total EphB4 protein amount, EphB4 was analyzed in the same cell lysates by western blot analysis (figure shows one representative blot out of three independent experiments performed in duplicate) ranging from 92% to 112% of control as calculated after densitometric analysis. Anti beta -actin served as loading control. (D) Immunohistochemical detection of EphB4 in acetone-fixed cryosections of A375 pIRES and A375 EphB4 tumor xenografts using goat anti EphB4 antibody. Sections stained without the primary antibody served as negative control. Scale bar 50 µm. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/29462967), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse EphB4 by Western Blot

Detection of Mouse EphB4 by Western Blot

EphB4 expression in transfected A375 melanoma cells and corresponding tumor xenografts. Western blot analysis of EphB4 and its ligand EphrinB2 in A375, A375-pIRES, and A375-EphB4 whole cell lysates (A) and tumor lysates (E). Anti-beta -actin served as loading control. (B) Relative mRNA expression of EphB4 and EphrinB2 in A375, A375-pIRES, and A375-EphB4 cells, analyzed by quantitative real-time RT-PCR was normalized to the constitutive expression level of beta -actin and to expression level in wild-type A375 melanoma cells using the delta delta CT method (2−∆∆ct) resulting in a value of 1 for A375 cells. Values represent mean ± SEM of at least three independent experiments each performed in triplicate (* p < 0.05, *** p < 0.001). (C) EphB4 phosphorylation was analyzed by pEphB4-ELISA in whole cell lysates of A375 EphB4 cells incubated with different concentrations of sEphrinB2-Fc (0; 0,5; 1 µg/mL) for 15, 30, and 60 min. Values represent mean ± SD from one of at least three independent experiments, each performed in duplicate. To rule out influence of sEphrinB2 on total EphB4 protein amount, EphB4 was analyzed in the same cell lysates by western blot analysis (figure shows one representative blot out of three independent experiments performed in duplicate) ranging from 92% to 112% of control as calculated after densitometric analysis. Anti beta -actin served as loading control. (D) Immunohistochemical detection of EphB4 in acetone-fixed cryosections of A375 pIRES and A375 EphB4 tumor xenografts using goat anti EphB4 antibody. Sections stained without the primary antibody served as negative control. Scale bar 50 µm. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/29462967), licensed under a CC-BY license. Not internally tested by R&D Systems.

Applications for Human EphB4 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: MCF‑7 human breast cancer cell line

Immunohistochemistry

5-15 µg/mL
Sample: Immersion fixed paraffin-embedded sections of human kidney

Knockout Validated

EphB4 is specifically detected in HEK293T human embryonic kidney parental cell line but is not detectable in EphB4 knockout HEK293T cell line.

Simple Western

50 µg/mL
Sample: HUVEC human umbilical vein endothelial cells

Western Blot

2 µg/mL
Sample: K562 human chronic myelogenous leukemia cell line, COLO 205 human colorectal adenocarcinoma cell line, ZR‑75 human breast cancer cell line, and HUVEC human umbilical vein endothelial cells

Reviewed Applications

Read 1 review rated 5 using AF3038 in the following applications:

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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: EphB4

EphB4, also known as Htk, Myk1, Tyro11, and Mdk2, is a member of the Eph receptor tyrosine kinase family and binds Ephrin-B2. The A and B class Eph proteins have a common structural organization (1-4). The human EphB4 cDNA encodes a 987 amino acid (aa) precursor that includes a 15 aa signal sequence, a 524 aa extracellular domain (ECD), a 21 aa transmembrane segment, and a 427 aa cytoplasmic domain (5). The ECD contains an N-terminal globular domain, a cysteine-rich domain, and two fibronectin type III domains. The cytoplasmic domain contains a juxtamembrane motif with two tyrosine residues which are the major autophosphorylation sites, a kinase domain, and a conserved sterile alpha motif (SAM) (5). Activation of kinase activity occurs after membrane-bound or clustered ligand recognition and binding. The ECD of human EphB4 shares 89% aa sequence identity with mouse EphB4 and 42-45% aa sequence identity with human EphB1, 2, and 3. EphB4 is expressed preferentially on venous endothelial cells (EC) and inhibits cell-cell adhesion, chemotaxis, and angiogenesis. Opposing effects are induced by signaling through Ephrin-B2 expressed on arterial EC: adhesion, endothelial cell migration, and vessel sprouting (6). EphB4 singaling contributes to new vascularization by guiding venous EC away from Ephrin-B2 expressing EC. Ephrin-B2 signaling induces arterial EC to migrate towards nascent EphB4 expressing vessels (6). The combination of forward signaling through EphB4 and reverse signaling through Ephrin-B2 promotes in vivo mammary tumor growth and
tumor-associated angiogenesis (7). EphB4 promotes the differentiation of megakaryocytic and erythroid progenitors but not granulocytic or monocytic progenitors (8, 9).

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. Bennett, B.D. et al. (1994) J. Biol. Chem. 269:14211.
  6. Fuller, T. et al. (2003) J. Cell Sci. 116:2461.
  7. Noren, N.K. et al. (2004) Proc. Natl. Acad. Sci. USA 101:5583.
  8. Wang, Z. et al. (2002) Blood 99:2740.
  9. Inada, T. et al. (1997) Blood 89:2757.

Long Name

Eph Receptor B4

Alternate Names

Htk, Mdk2, Myk1, Tyro11

Entrez Gene IDs

2050 (Human); 13846 (Mouse)

Gene Symbol

EPHB4

UniProt

P54760

Additional EphB4 Products

Product Documents for Human EphB4 Antibody

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Product Specific Notices for Human EphB4 Antibody

For research use only

Citations for Human EphB4 Antibody

Customer Reviews for Human EphB4 Antibody (1)

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  • Human EphB4 Antibody
    Name: Anonymous
    Application: ELISA
    Sample Tested: Serum and Plasma
    Species: Human
    Verified Customer | Posted 07/05/2022
    works well for ELISA in combination with mAb
    Human EphB4 Antibody AF3038

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