Key Product Details

Validated by

Biological Validation

Species Reactivity

Validated:

Mouse

Cited:

Human, Mouse, Rat, Fish - Danio rerio (Zebrafish), Primate - Chlorocebus aethiops (African Green Monkey)

Applications

Validated:

Immunohistochemistry, Western Blot, Flow Cytometry, CyTOF-ready

Cited:

Immunohistochemistry, Immunohistochemistry-Paraffin, Immunohistochemistry-Frozen, Western Blot, Flow Cytometry, Immunofluorescence, Immunoprecipitation, Bioassay

Label

Unconjugated

Antibody Source

Polyclonal Goat IgG
View all EphB4 Primary Antibodies »

Product Specifications

Immunogen

Mouse myeloma cell line NS0-derived recombinant mouse EphB4
Leu16-Ala539
Accession # P54761

Specificity

Detects mouse EphB4 in direct ELISAs and Western blots. In direct ELISAs, approximately 5% cross-reactivity with recombinant mouse (rm) EphB6 and rmEphA3 is observed and less than 1% cross-reactivity with rmEphA2, rmEphA4, rmEphA6, rmEphA7, rmEphA8, rmEphB2, and rmEphB3 is observed.

Clonality

Polyclonal

Host

Goat

Isotype

IgG

Scientific Data Images for Mouse EphB4 Antibody

EphB4 antibody in Mouse Embryo by Immunohistochemistry (IHC-Fr).

EphB4 in Mouse Embryo.

EphB4 was detected in immersion fixed frozen sections of mouse embryo (15 d.p.c.) using Goat Anti-Mouse EphB4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF446) at 15 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). View our protocol for Chromogenic IHC Staining of Frozen Tissue Sections.

Detection of Mouse EphB4 by Western Blot

Detection of Mouse EphB4 by Western Blot

Reduced Eph-B4 activity increases venous neointimal thickening. (A) Representative photomicrographs (left panel) and bar graph (right panel) showing AVF venous limb wall thickness in control and Eph-B4 het mice (day 21); *P = 0.047 (t-test). n = 8. Scale bar 25 µm. (B) Line graph showing infrarenal IVC diameter in control or Eph-B4 het mice; *P = 0.59 (ANOVA). n = 8–9. (C) Representative Western blot showing inhibited tyrosine phosphorylation in the Y774F-Eph-B4 mutant compared to the WT-Eph-B4 construct (0–60 min). (D) Bar graph showing Ephrin-B2/Fc stimulated COS cell migration after transfection with WT-Eph-B4 or Y774F-Eph-B4 plasmids. P < 0.0001 (ANOVA); *P < 0.0001 Ephrin-B2/Fc WT-Eph-B4 vs Y774F-Eph-B4. n = 3–4. (E) Representative photomicrographs (left panel) showing AVF venous wall (elastin stain) in control mice or mice treated with WT-Eph-B4 or mutant Y774F-Eph-B4. Arrow heads denote neointimal thickness. Scale bar, 25 µm. Bar graph (right panel) showing quantification of AVF venous wall thickness in control mice (white bar) or mice treated with WT-Eph-B4 (gray bar) or mutant Y774F-Eph-B4 (blue bar), day 21; P = 0.035 (ANOVA). *P = 0.038 (WT-Eph-B4 vs Y774F-Eph-B4; post hoc). n = 5–7. (F) Line graph showing infrarenal IVC diameter in mice with AVF treated with WT-Eph-B4 (gray line) or mutant Y774F-Eph-B4 (purple line) compared to control (black line); *P = 0.005 (ANOVA). n = 5–11. Data represent mean ± SEM. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/29133876), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Human EphB4 by Western Blot

Detection of Human EphB4 by Western Blot

Increased Eph-B4 and Ephrin-B2 expression during adaptive venous remodeling. (A) Western blot and adjacent bar graph of densitometry showing human Eph-B4 expression in AVF venous limb compared to normal vein. *P = 0.0016; t-test. n = 3–4. (B) Line graphs show expression of Eph-B4 (blue) and Ephrin-B2 (red) in the AVF venous limb compared to sham IVC; P < 0.0001 (ANOVA). *P < 0.05 (P = 0.0123, Eph-B4; P = 0.0041, Ephrin-B2; post hoc); **P < 0.05 (P < 0.0001, Ephrin-B2; post hoc). n = 5–8. (C) Western blots showing Eph-B4 and Ephrin-B2 protein expression in AVF venous limb compared to sham IVC. n = 3–5. (D) Graphs showing densitometry of Eph-B4 (left panel) and Ephrin-B2 (right panel) expression in the AVF venous limb compared to sham IVC; *P < 0.05 (P < 0.0001, Eph-B4 day 7, AVF vs sham; P < 0.0001, Eph-B4 day 21, AVF vs sham; P < 0.0001, Ephrin-B2 day 7, AVF vs sham; post hoc). n = 3–5. (E) Diagram of rat model showing location of infrarenal IVC pericardial patch exposed to an aortocaval AVF (n = 6 per group). (F) Representative Western blot (upper panel) showing Eph-B4 and Ephrin-B2 expression in patch neointima (day 14) of control vein compared to patch neointima of AVF vein. Graphs (lower panel) show quantification of western blot bands; P < 0.0001 (ANOVA). *P < 0.05 (P = 0.0003, Eph-B4; P = 0.0043, Ephrin-B2; post hoc). n = 3. (G) Representative photomicrographs (upper panel) showing Eph-B4 (green) and Ephrin-B2 (red) immunoreactive signal (day 14). White arrowheads indicate colocalization of Eph-B4 and Ephrin-B2. L, vessel lumen. Graph (lower panel) shows quantification of immunoreactive signal; P < 0.0001 (ANOVA). *P < 0.05 (P = 0.0136 Eph-B4; P < 0.0001 Ephrin-B2; post hoc). n = 3. Scale bar 100 µm. Data represent mean ± SEM. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/29133876), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Human EphB4 by Western Blot

Detection of Human EphB4 by Western Blot

Increased Eph-B4 and Ephrin-B2 expression during adaptive venous remodeling. (A) Western blot and adjacent bar graph of densitometry showing human Eph-B4 expression in AVF venous limb compared to normal vein. *P = 0.0016; t-test. n = 3–4. (B) Line graphs show expression of Eph-B4 (blue) and Ephrin-B2 (red) in the AVF venous limb compared to sham IVC; P < 0.0001 (ANOVA). *P < 0.05 (P = 0.0123, Eph-B4; P = 0.0041, Ephrin-B2; post hoc); **P < 0.05 (P < 0.0001, Ephrin-B2; post hoc). n = 5–8. (C) Western blots showing Eph-B4 and Ephrin-B2 protein expression in AVF venous limb compared to sham IVC. n = 3–5. (D) Graphs showing densitometry of Eph-B4 (left panel) and Ephrin-B2 (right panel) expression in the AVF venous limb compared to sham IVC; *P < 0.05 (P < 0.0001, Eph-B4 day 7, AVF vs sham; P < 0.0001, Eph-B4 day 21, AVF vs sham; P < 0.0001, Ephrin-B2 day 7, AVF vs sham; post hoc). n = 3–5. (E) Diagram of rat model showing location of infrarenal IVC pericardial patch exposed to an aortocaval AVF (n = 6 per group). (F) Representative Western blot (upper panel) showing Eph-B4 and Ephrin-B2 expression in patch neointima (day 14) of control vein compared to patch neointima of AVF vein. Graphs (lower panel) show quantification of western blot bands; P < 0.0001 (ANOVA). *P < 0.05 (P = 0.0003, Eph-B4; P = 0.0043, Ephrin-B2; post hoc). n = 3. (G) Representative photomicrographs (upper panel) showing Eph-B4 (green) and Ephrin-B2 (red) immunoreactive signal (day 14). White arrowheads indicate colocalization of Eph-B4 and Ephrin-B2. L, vessel lumen. Graph (lower panel) shows quantification of immunoreactive signal; P < 0.0001 (ANOVA). *P < 0.05 (P = 0.0136 Eph-B4; P < 0.0001 Ephrin-B2; post hoc). n = 3. Scale bar 100 µm. Data represent mean ± SEM. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/29133876), licensed under a CC-BY license. Not internally tested by R&D Systems.

Applications for Mouse 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

2.5 µg/106 cells
Sample: MCF‑7 human breast cancer cell line

Immunohistochemistry

5-15 µg/mL
Sample: Immersion fixed frozen sections of mouse embryo (E13-15)

Western Blot

0.1 µg/mL
Sample: Recombinant Mouse EphB4 Fc Chimera (Catalog # 446-B4)

Reviewed Applications

Read 4 reviews rated 4.3 using AF446 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: EphB4

EphB4, also known as Htk, Myk1, Tyro11, and Mdk2 (1), is a member of the Eph receptor family which binds members of the ephrin ligand family. There are two classes of receptors, designated A and B. Both the A and B class receptors have an extracellular region consisting of a globular domain, a cysteine-rich domain, and two fibronectin type III domains. This is followed by the transmembrane region and cytoplasmic region. The cytoplasmic region contains a juxtamembrane motif with two tyrosine residues, which are the major autophosphorylation sites, a kinase domain, and a conserved sterile alpha motif (SAM) in the carboxy tail which contains one conserved tyrosine residue. Activation of kinase activity occurs after ligand recognition and binding. EphB4 has been shown to bind ephrin‑B2 and ephrin‑B1 (2, 3). The extracellular domains of human and mouse EphB4 share 88% amino acid identity. Only membrane-bound or Fc-clustered ligands are capable of activating the receptor in vitro. While soluble monomeric ligands bind the receptor, they do not induce receptor autophosphorylation and activation (2). In vivo, the ligands and receptors display reciprocal expression (3). It has been found that nearly all receptors and ligands are expressed in developing and adult neural tissue (3). The Eph/ephrin families also appear to play a role in angiogenesis (3).

References

  1. Eph Nomenclature Committee [letter] (1997) Cell 90:403.
  2. Flanagan, J.G. and P. Vanderhaeghen (1998) Annu. Rev. Neurosci. 21:309.
  3. Pasquale, E.B. (1997) Curr. Opin. Cell Biol. 9:608.

Long Name

Eph Receptor B4

Alternate Names

Htk, Mdk2, Myk1, Tyro11

Entrez Gene IDs

2050 (Human); 13846 (Mouse)

Gene Symbol

EPHB4

UniProt

P54761

Additional EphB4 Products

Product Documents for Mouse EphB4 Antibody

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Product Specific Notices for Mouse EphB4 Antibody

For research use only

Citations for Mouse EphB4 Antibody

Customer Reviews for Mouse EphB4 Antibody (4)

4.3 out of 5
4 Customer Ratings
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Showing  1 - 4 of 4 reviews Showing All
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  • Mouse EphB4 Antibody
    Name: Anonymous
    Application: Immunocytochemistry/Immunofluorescence
    Sample Tested: E12.5 mouse embryo fixed in 4% PFA
    Species: Mouse
    Verified Customer | Posted 12/16/2020
    Dilution used - 1:200. The staining was done on an E12.5 transverse (4% PFA fixed) mouse section using standard IF techniques. Used 1% BSA for blocking for 1 hour before adding the primary antibody. The results are hit or miss for me, because I have noticed expression on non target tissue (Sometimes it doesn't appear, sometimes it does). Attached pictures shows a artery (smaller circle) and a vein (bigger circle), and while the EphB4 expression on the vein is very good (as it is supposed to express there), there seems to be some non-specific expression on the adjacent artery as well.
    Mouse EphB4 Antibody AF446
  • Mouse EphB4 Antibody
    Name: Anonymous
    Application: Western Blot
    Sample Tested: embryos
    Species: Zebrafish
    Verified Customer | Posted 11/13/2020
    Mouse EphB4 Antibody AF446
  • Name: Anonymous
    Application: Western Blot
    Sample Tested: See PMID 22398409
    Species: Rat
    Verified Customer | Posted 01/08/2015
  • Name: Anonymous
    Application: Immunofluorescence
    Sample Tested: See PMID 22398409
    Species: Rat
    Verified Customer | Posted 01/08/2015

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