Overview

  • Product name
  • Description
    Rabbit polyclonal to Ephrin B2
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, ICC/IFmore details
  • Species reactivity
    Reacts with: Human
    Predicted to work with: Rat
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 150 - 250 of Human Ephrin B2.

    Read Abcam's proprietary immunogen policy

  • Positive control
    • This antibody gave a positive signal in Human lung and Human kidney tissue lysates.

Properties

Applications

Our Abpromise guarantee covers the use of ab96264 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB Use a concentration of 1 µg/ml. Detects a band of approximately 32 kDa (predicted molecular weight: 36 kDa).
ICC/IF Use a concentration of 5 µg/ml.

Target

  • Function
    Cell surface transmembrane ligand for Eph receptors, a family of receptor tyrosine kinases which are crucial for migration, repulsion and adhesion during neuronal, vascular and epithelial development. Binds promiscuously Eph receptors residing on adjacent cells, leading to contact-dependent bidirectional signaling into neighboring cells. The signaling pathway downstream of the receptor is referred to as forward signaling while the signaling pathway downstream of the ephrin ligand is referred to as reverse signaling. Binds to receptor tyrosine kinase including EPHA4, EPHA3 and EPHB4. Together with EPHB4 plays a central role in heart morphogenesis and angiogenesis through regulation of cell adhesion and cell migration. EPHB4-mediated forward signaling controls cellular repulsion and segregation from EFNB2-expressing cells. May play a role in constraining the orientation of longitudinally projecting axons.
    (Microbial infection) Acts as a receptor for Hendra virus and Nipah virus.
  • Tissue specificity
    Lung and kidney.
  • Sequence similarities
    Belongs to the ephrin family.
    Contains 1 ephrin RBD (ephrin receptor-binding) domain.
  • Post-translational
    modifications
    Inducible phosphorylation of tyrosine residues in the cytoplasmic domain.
  • Cellular localization
    Membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • EFN B2 antibody
    • EFNB 2 antibody
    • Efnb2 antibody
    • EFNB2_HUMAN antibody
    • Eph related receptor tyrosine kinase ligand 5 antibody
    • EPH-related receptor tyrosine kinase ligand 5 antibody
    • ephrin B2 antibody
    • Ephrin-B2 antibody
    • EphrinB2 antibody
    • EPLG 5 antibody
    • EPLG5 antibody
    • Htk L antibody
    • HTK ligand antibody
    • HTK-L antibody
    • HTKL antibody
    • LERK 5 antibody
    • LERK-5 antibody
    • LERK5 antibody
    • Ligand of eph related kinase 5 antibody
    • MGC126226 antibody
    • MGC126227 antibody
    • MGC126228 antibody
    • OTTMUSP00000024973 antibody
    see all

Images

  • All lanes : Anti-Ephrin B2 antibody (ab96264) at 1 µg/ml

    Lane 1 : Lung (Human) Tissue Lysate
    Lane 2 : Human kidney tissue lysate - total protein (ab30203)

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 36 kDa
    Observed band size: 32 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 73 kDa, 86 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 12 minutes


    The band observed at 32 kDa could potentially be a cleaved form of Ephrin B2 due to the presence of a 27 amino acid signal peptide.
  • ICC/IF image of ab96264 stained MCF7 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab96264, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

References

This product has been referenced in:
  • Sivarapatna A  et al. Engineered Microvasculature in PDMS Networks Using Endothelial Cells Derived from Human Induced Pluripotent Stem Cells. Cell Transplant 26:1365-1379 (2017). Read more (PubMed: 28901188) »
  • Alam SK  et al. DNA damage-induced ephrin-B2 reverse signaling promotes chemoresistance and drives EMT in colorectal carcinoma harboring mutant p53. Cell Death Differ 23:707-22 (2016). Read more (PubMed: 26494468) »
See all 2 Publications for this product

Customer reviews and Q&As

1-5 of 5 Abreviews or Q&A

Answer

Thank you for your reply.

I am happy to hear the staining has improved!

The antibody was raised against an extracellular sequence, but since it is a polyclonal it really could have multiple binding epitopes throughout the protein and perhaps a region does correspond to an intracellular portion.

We ha found the best results with permeabilization as well, so it is great to hear that you are seeing consistent staining now.

Please contact us with any other questions.

Read More

Answer

Thank you for your reply.

Yes, I think you should include the blocking buffer in with the secondary. Typically we recommend 1% BSA, but 5% BSA should be okay.

https://www.abcam.com/index.html?pageconfig=resource&rid=14733

Let me know if the staining doesn't improve. I hope this information helps.

Read More

Answer

Thank you for taking time to contact us. I am sorry to hear that this antibody is not providing satisfactory results.

So the antibody is staining well in immunocytochemistry (ICC/IF)on fixed human endothelial cells? What is the fixative you're using for these?

And the antibody is not working well on frozen tissue sections (IHC-Fr)? What type of tissue are you staining? How thick are the sections? How are they fixed (i.e. methanol)?

Do you have images of the IHC-Fr sections and ICC/IF cells?

Lung and kidney should be good positive control tissues, but I don't know of any negative control tissues. You can take a look at the Human Protein Atlas as tonsilnon-germinal center cells as these didn't show any positive staining. http://www.proteinatlas.org/ENSG00000125266/normal

If you are seeing background in your no-primary control, it means that your secondary is binding non-specifically. You may want to dilute the secondary more, include blocking buffer in with the secondary, or switch to a secondary that is pre-adsorbed to remove cross-reactivity with human IgG.

We haven't tested this antibody in IHC-Fr, only in ICC/IF. This is the protocol used: ICC/IF image of ab96264 stained MCF7 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab96264, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Should the suggestions not improve the results, please do let me know.

I hope this informationhelps. Please contact us with any other questions.

Read More

Answer

Let me just confirm it is the products Anti-Ephrin B2 antibody (ab96264) and Anti-Ephrin B2 (phospho Y316) antibody (ab119323) which you are interested in?

The anti-Ephrin B2 antibody (ab96264) does indeed measure the total Ephrin B2 content, the Y316 phosphorylated as well as unphosphorylated form of the protein. This is in contrast to the anti-Ephrin B2 (phospho Y316) antibody (ab119323) which specifically detects the Y316 phosphorylated form of the protein. The ab96264 could indeed be used as a control when using the ab119323 to detect the total protein present in the samples.

I hope this information has been of help. If you require any further information please do not hesitate to ask.

Have a lovely weekend!

Read More

Question
Answer

Thank you for calling Abcam earlier today.

I am sorry about the problems you were having with ab96264 in IF. As we discussed, I am sending you a new vial of the antibody to test. Your new order/reference number is *******.

If there is anything else I can help you with, please let me know.

Have a great weekend.

Read More

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"
For licensing inquiries, please contact partnerships@abcam.com

Sign up