Astana Biomed Group, an authorized Abcam distributor in Central Asia

Anti-KAP1 antibody [EPR5249] (ab109545)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR5249] to KAP1
  • Suitable for: IP, Flow Cyt, WB, IHC-P, ICC/IF
  • Knockout validated
  • Reacts with: Mouse, Human

Overview

  • Product name

    Anti-KAP1 antibody [EPR5249]
    See all KAP1 primary antibodies

  • Description

    Rabbit monoclonal [EPR5249] to KAP1

  • Host species

    Rabbit

  • Specificity

    Although the immunogen is from a phosphor-peptide, this antibody detects phospho and non-phospho KAP1. Based on a peptide blocking experiment it has been found that the signal generated after non-phospho peptide blocking became much weaker, thus indicating that ab109545 shows cross-reactivity with the non-phospho KAP1 at high level.

  • Tested Applications & Species

    Application Species
    Flow Cyt
    Human
    ICC/IF
    Human
    IHC-P
    Human
    IP
    Human
    WB
    Human
    See all applications and species data

  • Immunogen

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

  • Positive control

    • WB: A431 and MCF7 cell lysates; IHC-P: Human colon and kidney tissue; ICC/IF: Hela cells; IP: A431 cell lysate.

  • 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-KAP1 antibody [EPR5249] (ab109545)
    Western blot - Anti-KAP1 antibody [EPR5249] (ab109545)

    Lane 1: Wild type HAP1 whole cell lysate (20 µg)
    Lane 2: HAP1 + DMSO whole cell lysate (20 µg)
    Lane 3: HAP1 + Blaomycin whole cell lysate (20 µg)
    Lane 4: TRIM28 knockout HAP1 whole cell lysate (20 µg)
    Lane 5: TRIM28 knockout HAP1 + DMSO whole cell lysate (20 µg)
    Lane 6: TRIM28 knockout HAP1 + Blaomycin whole cell lysate (20 µg)
    Lane 7: HeLa + DMSO whole cell lysate (20 µg)
    Lane 8: HeLa + Blaomycin whole cell lysate (20 µg)

    Lanes 1 - 8: Merged signal (red and green). Green - ab109545 observed at 110 kDa. Red - loading control, ab8245, observed at 37 kDa.

    ab109545 was shown to specifically react with KAP1 in wild type cells as signal was lost in KAP1 knockout cells. Wild-type and KAP1 knockout samples were subjected to SDS-PAGE. ab109545 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 10000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

  • Immunocytochemistry/ Immunofluorescence - Anti-KAP1 antibody [EPR5249] (ab109545)
    Immunocytochemistry/ Immunofluorescence - Anti-KAP1 antibody [EPR5249] (ab109545)
    ab109545, at a 1/100 dilution, staining KAP1 in HeLa cells
  • Immunoprecipitation - Anti-KAP1 antibody [EPR5249] (ab109545)
    Immunoprecipitation - Anti-KAP1 antibody [EPR5249] (ab109545)
    KAP1 was immunoprecipitated from 0.35 mg A431 (Human epidermoid carcinoma epithelial cell) cell lysate 10 µg with ab109545 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab109545 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

    Lane 1: A431 (Human epidermoid carcinoma epithelial cell) cell lysate 10 µg

    Lane 2: ab109545 IP in A431 cell lysate

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab109545 in A431 cell lysate

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

    Exposure time: 32 seconds

  • Western blot - Anti-KAP1 antibody [EPR5249] (ab109545)
    Western blot - Anti-KAP1 antibody [EPR5249] (ab109545)
    All lanes : Anti-KAP1 antibody [EPR5249] (ab109545) at 1/10000 dilution

    Lane 1 : A431 (Human epidermoid carcinoma epithelial cell) whole cell lysates
    Lane 2 : MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates

    Lysates/proteins at 15 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

    Developed using the ECL technique.

    Predicted band size: 89 kDa
    Observed band size: 110 kDa
    why is the actual band size different from the predicted?


    Exposure time: 10 seconds


    Blocking/diluting buffer and concentration: 5% NFDM/TBST

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KAP1 antibody [EPR5249] (ab109545)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KAP1 antibody [EPR5249] (ab109545)

    ab109545, at a 1/100 dilution, staining KAP1 in formalin-fixed, paraffin-embedded Human colon tissue.

    Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KAP1 antibody [EPR5249] (ab109545)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KAP1 antibody [EPR5249] (ab109545)

    ab109545, at a 1/100 dilution, staining KAP1 in formalin-fixed, paraffin-embedded Human kidney tissue.

    Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

  • Flow Cytometry - Anti-KAP1 antibody [EPR5249] (ab109545)
    Flow Cytometry - Anti-KAP1 antibody [EPR5249] (ab109545)
    Overlay histogram showing HeLa cells stained with ab109545 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab190545, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • Anti-KAP1 antibody [EPR5249] (ab109545)
    Anti-KAP1 antibody [EPR5249] (ab109545)

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