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    eph-receptor-b4htk-antibody-epr23221-54-ab254300.pdf

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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-Eph receptor B4/HTK antibody [EPR23221-54] (ab254300)

  • Datasheet
  • Certificate of Compliance
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Western blot - Anti-Eph receptor B4/HTK antibody [EPR23221-54] (ab254300)
  • Immunoprecipitation - Anti-Eph receptor B4/HTK antibody [EPR23221-54] (ab254300)
  • Western blot - Anti-Eph receptor B4/HTK antibody [EPR23221-54] (ab254300)
  • Immunoprecipitation - Anti-Eph receptor B4/HTK antibody [EPR23221-54] (ab254300)
  • Anti-Eph receptor B4/HTK antibody [EPR23221-54] (ab254300)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR23221-54] to Eph receptor B4/HTK
  • Suitable for: WB, IP
  • Knockout validated
  • Reacts with: Human

Conjugates logo Related conjugates and formulations

Carrier Free

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Knockout
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Human EPHB4 (Eph receptor B4/HTK) knockout HEK-293T cell line (ab266733)

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Overview

  • Product name

    Anti-Eph receptor B4/HTK antibody [EPR23221-54]
    See all Eph receptor B4/HTK primary antibodies
  • Description

    Rabbit monoclonal [EPR23221-54] to Eph receptor B4/HTK
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IPmore details
    Unsuitable for: Flow Cyt,ICC/IF or IHC-P
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: HEK293T, HT-29, HUVEC, HCT 116 and MCF7 lysates. IP: HT-29 and HUVEC whole cell lysates.
  • General notes

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
  • Storage buffer

    pH: 7.2
    Preservative: 0.01% Sodium azide
    Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EPR23221-54
  • Isotype

    IgG
  • Research areas

    • Cardiovascular
    • Angiogenesis
    • Ephrins
    • Eph Receptors
    • Neuroscience
    • Neurotransmission
    • Intracellular Signaling
    • Kinases
    • Neuroscience
    • Cell Type Marker
    • Neuron marker
    • Growth Cone
    • Neuroscience
    • Neurology process
    • Growth and Development
    • Axonal Guidance Proteins
    • Neuroscience
    • Neurotransmission
    • Receptors / Channels
    • Tyrosine Kinase Receptors
    • Stem Cells
    • Hematopoietic Progenitors
    • Surface Molecules

Associated products

  • Alternative Versions

    • Anti-Eph receptor B4/HTK antibody [EPR23221-54] - BSA and Azide free (ab269867)
  • Compatible Secondaries

    • VeriBlot for IP Detection Reagent (HRP) (ab131366)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab97051)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • KO cell lines

    • Human EPHB4 (Eph receptor B4/HTK) knockout HEK-293T cell line (ab266733)
  • KO cell lysates

    • Human EPHB4 (Eph receptor B4/HTK) knockout HEK-293T cell lysate (ab257429)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab254300 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
1/1000. Detects a band of approximately 120 kDa (predicted molecular weight: 108 kDa).
IP
1/30.
Notes
WB
1/1000. Detects a band of approximately 120 kDa (predicted molecular weight: 108 kDa).
IP
1/30.
Application notes
Is unsuitable for Flow Cyt,ICC/IF or IHC-P.

Target

  • Function

    Receptor for members of the ephrin-B family. Binds to ephrin-B2. May have a role in events mediating differentiation and development.
  • Tissue specificity

    Abundantly expressed in placenta and in a range of primary tissues and malignant cell lines. Expressed in fetal, but not adult, brain, and in primitive and myeloid, but not lymphoid, hematopoietic cells.
  • Sequence similarities

    Belongs to the protein kinase superfamily. Tyr protein kinase family. Ephrin receptor subfamily.
    Contains 2 fibronectin type-III domains.
    Contains 1 protein kinase domain.
    Contains 1 SAM (sterile alpha motif) domain.
  • Post-translational
    modifications

    Autophosphorylated.
  • Cellular localization

    Membrane.
  • Target information above from: UniProt accession P54760 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 2050 Human
    • Omim: 600011 Human
    • SwissProt: P54760 Human
    • Unigene: 437008 Human
    • Alternative names

      • Ephb4 antibody
      • EPHB4_HUMAN antibody
      • Ephrin receptor EphB4 antibody
      • Ephrin type-B receptor 4 antibody
      • Hepatoma Transmembrane Kinase antibody
      • HTK antibody
      • MYK 1 antibody
      • MYK1 antibody
      • TYRO 11 antibody
      • TYRO11 antibody
      • Tyrosine-protein kinase receptor HTK antibody
      • Tyrosine-protein kinase TYRO11 antibody
      see all

    Images

    • Western blot - Anti-Eph receptor B4/HTK antibody [EPR23221-54] (ab254300)
      Western blot - Anti-Eph receptor B4/HTK antibody [EPR23221-54] (ab254300)
      All lanes : Anti-Eph receptor B4/HTK antibody [EPR23221-54] (ab254300) at 1/1000 dilution

      Lane 1 : Wild-type HEK293T cell lysate
      Lane 2 : EPHB4 knockout HEK293T cell lysate
      Lane 3 : MCF7 cell lysate
      Lane 4 : HT-29 cell lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

      Predicted band size: 108 kDa
      Observed band size: 125 kDa why is the actual band size different from the predicted?



      Lanes 1-4: Merged signal (red and green). Green - ab254300 observed at 125 kDa. Red - loading control ab8245 observed at 36 kDa.

       ab254300 Anti-Eph receptor B4/HTK antibody [EPR23221-54] was shown to specifically react with Eph receptor B4/HTK in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266733 (knockout cell lysate ab257429) was used. Wild-type and Eph receptor B4/HTK knockout samples were subjected to SDS-PAGE. ab254300 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

    • Immunoprecipitation - Anti-Eph receptor B4/HTK antibody [EPR23221-54] (ab254300)
      Immunoprecipitation - Anti-Eph receptor B4/HTK antibody [EPR23221-54] (ab254300)

      Eph receptor B4 was immunoprecipitated from 0.35 mg HUVEC (human umbilical vein endothelial cell) whole cell lysate with ab254300 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab254300 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.

      Lane 1: HUVEC (human umbilical vein endothelial cell) whole cell lysate 10ug

      Lane 2: ab254300 IP in HUVEC whole cell lysate

      Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254300 in HUVEC whole cell lysate

      Blocking and dilution buffer and concentration: 5% NFDM/TBST.

      Exposure time: 8 seconds.

      Cleaved intracellular domain (ICD) was observed at around 47kDa. (PMID: 24854540).

    • Western blot - Anti-Eph receptor B4/HTK antibody [EPR23221-54] (ab254300)
      Western blot - Anti-Eph receptor B4/HTK antibody [EPR23221-54] (ab254300)
      All lanes : Anti-Eph receptor B4/HTK antibody [EPR23221-54] (ab254300) at 1/1000 dilution

      Lane 1 : HT-29 (human colorectal adenocarcinoma epithelial cell), whole cell lysate
      Lane 2 : HUVEC (human umbilical vein endothelial cell), whole cell lysate
      Lane 3 : SW620 (human colorectal adenocarcinoma epithelial cell), whole cell lysate
      Lane 4 : HCT 116 (human colorectal carcinoma epithelial cell), whole cell lysate
      Lane 5 : MCF7 (human breast adenocarcinoma epithelial cell), whole cell lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

      Predicted band size: 108 kDa
      Observed band size: 120 kDa why is the actual band size different from the predicted?



      Blocking and dilution buffer: 5% NFDM/TBST.

      Exposure time: 37 seconds.

      Negative control: SW620 (PMID: 19366806).

      EphB4 is negative in SW620 and positive in HT29 and HUVEC, which is different from expression profiles of other EphB family members.

      Cleaved intracellular domain (ICD) was observed at around 47kDa. (PMID: 24854540).

      The expression profile observed is consistent with what has been described in the literature (PMID: 19366806, 16840724).

    • Immunoprecipitation - Anti-Eph receptor B4/HTK antibody [EPR23221-54] (ab254300)
      Immunoprecipitation - Anti-Eph receptor B4/HTK antibody [EPR23221-54] (ab254300)

      Eph receptor B4 was immunoprecipitated from 0.35 mg HT-29 (human colorectal adenocarcinoma epithelial cell) whole cell lysate with ab254300 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab254300 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.

      Lane 1: HT-29 (human colorectal adenocarcinoma epithelial cell) whole cell lysate 10ug

      Lane 2: ab254300 IP in HT-29 whole cell lysate

      Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254300 in HT-29 whole cell lysate

      Blocking and dilution buffer and concentration: 5% NFDM/TBST.

      Exposure time: 8 seconds.

      Cleaved intracellular domain (ICD) was observed at around 47kDa. (PMID: 24854540).

    • Anti-Eph receptor B4/HTK antibody [EPR23221-54] (ab254300)
      Anti-Eph receptor B4/HTK antibody [EPR23221-54] (ab254300)

    Protocols

    To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

    Click here to view the general protocols

    Datasheets and documents

    • Datasheet download

      Download

    Certificate of Compliance

    To download a Certificate of Compliance, please enter your Lot number below:

    References (0)

    Publishing research using ab254300? Please let us know so that we can cite the reference in this datasheet.

    ab254300 has not yet been referenced specifically in any publications.

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