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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

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 & Species

    Application Species
    IP
    Human
    WB
    Human
    See all applications and species data

  • 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

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)

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