Astana Biomed Group, an authorized Abcam distributor in Central Asia

Anti-IKK beta antibody [Y466] (ab32135)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [Y466] to IKK beta
  • Suitable for: WB, IP
  • Knockout validated
  • Reacts with: Mouse, Human

Overview

  • Product name

    Anti-IKK beta antibody [Y466]
    See all IKK beta primary antibodies

  • Description

    Rabbit monoclonal [Y466] to IKK beta

  • Host species

    Rabbit

  • Tested Applications & Species

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

  • Immunogen

    Synthetic peptide within Human IKK beta aa 650-750 (C terminal). The exact sequence is proprietary.
    Database link: O14920

  • Positive control

    • WB: Wild-type HAP1 cell lysate. HeLa, NIH/3T3 and Daudi cell lysate. IHC-P: Human cervical carcinoma tissue. IP: SH-SY5Y whole cell lysate.

  • General notes

    Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

     

    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.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

Properties

Images

  • Western blot - Anti-IKK beta antibody [Y466] (ab32135)
    Western blot - Anti-IKK beta antibody [Y466] (ab32135)
    All lanes : Anti-IKK beta antibody [Y466] (ab32135) at 1/1000 dilution

    Lane 1 : Wild-type HeLa cell lysate
    Lane 2 : IKBKB knockout HeLa cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Observed band size: 87 kDa
    why is the actual band size different from the predicted?



    Lanes 1- 2: Merged signal (red and green). Green - ab32135 observed at 87 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

     ab32135 was shown to react with IKK beta in wild-type HeLa cells in western blot. The band observed in knockout cell line ab264847 (knockout cell lysate ab257228) lane below 87kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and IKBKB knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab32135 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 1000 dilution and a 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-IKK beta antibody [Y466] (ab32135)
    Immunoprecipitation - Anti-IKK beta antibody [Y466] (ab32135)

    ab32135 (purified) at 1:50 dilution (2μg) immunoprecipitating IKK beta in SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysate.

    Lane 1 (input): SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysate 10ug
    Lane 2 (+): ab32135 & SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysate
    Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32135 in SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysate

    For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:10,000 dilution.
    Blocking and diluting buffer: 5% NFDM/TBST.

  • Western blot - Anti-IKK beta antibody [Y466] (ab32135)
    Western blot - Anti-IKK beta antibody [Y466] (ab32135)

    Lanes 1, 3 and 5: Wild-type HAP1 cell lysate (20 µg)
    Lanes 2, 4 and 6: IKK beta knockout HAP1 cell lysate (20 µg)
    Lanes 1 and 2: Green signal from target – ab32135 observed at 87 kDa
    Lanes 3 and 4: Red signal from loading control – ab8226 observed at 42 kDa
    Lanes 5 and 6: Merged (red and green) signal

    Unpurified ab32135 was shown to react with IKK beta when IKK beta knockout samples were used, along with additional cross-reactive bands. Wild-type and IKK beta knockout samples were subjected to SDS-PAGE. ab32135 and ab8226 (loading control to beta actin) were diluted 1/1000 and 1/2000 respectively and incubated overnight at 4ºC. 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 1/10 000 dilution for 1 h at room temperature before imaging.

  • Western blot - Anti-IKK beta antibody [Y466] (ab32135)
    Western blot - Anti-IKK beta antibody [Y466] (ab32135)
    All lanes : Anti-IKK beta antibody [Y466] (ab32135) at 1/1000 dilution (purified)

    Lane 1 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates
    Lane 2 : Mouse lung lysates
    Lane 3 : Rat brain lysates
    Lane 4 : SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysates

    Lysates/proteins at 20 µg per lane.

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

    Observed band size: 87 kDa why is the actual band size different from the predicted?



    Blocking and diluting buffer: 5% NFDM/TBST

     

  • Western blot - Anti-IKK beta antibody [Y466] (ab32135)
    Western blot - Anti-IKK beta antibody [Y466] (ab32135)
    Anti-IKK beta antibody [Y466] (ab32135) at 1/2500 dilution (purified) + HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 20 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution


    Blocking and diluting buffer: 5% NFDM/TBST

  • Western blot - Anti-IKK beta antibody [Y466] (ab32135)
    Western blot - Anti-IKK beta antibody [Y466] (ab32135)
    Anti-IKK beta antibody [Y466] (ab32135) at 1/5000 dilution (unpurified) + Daudi cell lysate

    Observed band size: 87 kDa why is the actual band size different from the predicted?

  • Western blot - Anti-IKK beta antibody [Y466] (ab32135)
    Western blot - Anti-IKK beta antibody [Y466] (ab32135)

    unpurified ab32135

  • Anti-IKK beta antibody [Y466] (ab32135)
    Anti-IKK beta antibody [Y466] (ab32135)

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