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Human EphB4 Antibody

Catalog #: AF3038
7 Citations Datasheet / COA / SDS
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AF3038
AF3038-SP
Detection of Human EphB4 by Western Blot.
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Product Details
Citations (7)
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Summary Data Examples Preparation and Storage Reconstitution Calculator Background Product Datasheets Related Research Areas

Human EphB4 Antibody Summary

Species Reactivity
Human
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.
Source
Polyclonal Goat IgG
Purification
Antigen Affinity-purified
Immunogen
Mouse myeloma cell line NS0-derived recombinant human EphB4
Leu16-Ala539
Accession # P54760
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.
Label
Unconjugated

Applications

Recommended Concentration
Sample
Western Blot
2 µg/mL
See below
Simple Western
50 µg/mL
See below
Flow Cytometry
0.25 µg/106 cells
See below
Immunohistochemistry
5-15 µg/mL
See below
CyTOF-ready
Ready to be labeled using established conjugation methods. No BSA or other carrier proteins that could interfere with conjugation.
 
Knockout Validated
EphB4 is specifically detected in HEK293T human embryonic kidney parental cell line but is not detectable in EphB4 knockout HEK293T cell line.
 

Please Note: Optimal dilutions should be determined by each laboratory for each application. General Protocols are available in the Technical Information section on our website.

Scientific Data

Western Blot Detection of Human EphB4 antibody by Western Blot. View Larger

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.

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

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.

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

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.

Simple Western Detection of Human EphB4 antibody by Simple Western<sup>TM</sup>. View Larger

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.

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

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.

Reconstitution Calculator

Reconstitution Calculator

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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Preparation and Storage

Reconstitution
Reconstitute at 0.2 mg/mL in sterile PBS.
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Shipping
The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below. *Small pack size (SP) is shipped with polar packs. Upon receipt, store it immediately at -20 to -70 °C
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.

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
Entrez Gene IDs
2050 (Human); 13846 (Mouse)
Alternate Names
EC 2.7.10; EC 2.7.10.1; EPH receptor B4; EphB4; ephrin type-B receptor 4; hepatoma transmembrane kinase; Htk; HTKephrin receptor EphB4; Mdk2; Myk1; soluble EPHB4 variant 1; soluble EPHB4 variant 2; soluble EPHB4 variant 3; Tyro11; Tyrosine-protein kinase receptor HTK; Tyrosine-protein kinase TYRO11

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Citations for Human EphB4 Antibody

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

7 Citations: Showing 1 - 7
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  1. Hypoxia Triggers the Intravasation of Clustered Circulating Tumor Cells
    Authors: C Donato, L Kunz, F Castro-Gin, A Paasinen-S, K Strittmatt, BM Szczerba, R Scherrer, N Di Maggio, W Heusermann, O Biehlmaier, C Beisel, M Vetter, C Rochlitz, WP Weber, A Banfi, T Schroeder, N Aceto
    Cell Rep, 2020-09-08;32(10):108105.
    Species: Human, Mouse
    Sample Types: Cell Lysates
    Applications: Western Blot
  2. Downregulation of proinflammatory cytokines in HTLV-1-infected T cells by Resveratrol
    J Exp Clin Cancer Res, 2016-07-22;35(1):118.
    Species: Human
    Sample Types: Cell Lysates
    Applications: Immunoprecipitation, Western Blot
  3. EPHB4 kinase-inactivating mutations cause autosomal dominant lymphatic-related hydrops fetalis
    J Clin Invest, 2016-07-11;0(0):.
    Species: Human
    Sample Types: Cell Lysates
    Applications: Immunoprecipitation, Western Blot
  4. Instruction of circulating endothelial progenitors in vitro towards specialized blood-brain barrier and arterial phenotypes.
    Authors: Boyer-Di Ponio J, El-Ayoubi F, Glacial F, Ganeshamoorthy K, Driancourt C, Godet M, Perriere N, Guillevic O, Couraud P, Uzan G
    PLoS ONE, 2014-01-02;9(1):e84179.
    Species: Human
    Sample Types: Whole Cells
    Applications: ICC
  5. BCR-ABL-independent and RAS / MAPK pathway-dependent form of imatinib resistance in Ph-positive acute lymphoblastic leukemia cell line with activation of EphB4.
    Authors: Suzuki M, Abe A, Imagama S, Nomura Y, Tanizaki R, Minami Y, Hayakawa F, Ito Y, Katsumi A, Yamamoto K, Emi N, Kiyoi H, Naoe T
    Eur. J. Haematol., 2009-11-28;84(3):229-38.
    Species: Human
    Sample Types: Cell Lysates
    Applications: Western Blot
  6. Soluble EphB4 inhibition of PDGF-induced RPE migration in vitro.
    Authors: He S, Kumar SR, Zhou P, Krasnoperov V, Ryan SJ, Gill PS, Hinton DR
    Invest. Ophthalmol. Vis. Sci., 2009-08-20;51(1):543-52.
    Species: Human
    Sample Types: Whole Tissue
    Applications: IHC-Fr
  7. Activation of the receptor EphB4 by its specific ligand ephrin B2 in human osteoarthritic subchondral bone osteoblasts.
    Authors: Kwan Tat S, Pelletier JP, Amiable N, Boileau C, Lajeunesse D, Duval N, Martel-Pelletier J
    Arthritis Rheum., 2008-12-01;58(12):3820-30.
    Species: Human
    Sample Types: Whole Tissue
    Applications: IHC

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