Anti-Topoisomerase II alpha + Topoisomerase II beta antibody [EPR5377] (ab109524)

Overview

Properties

Applications

Our Abpromise guarantee covers the use of ab109524 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
ICC/IF Use a concentration of 10 µg/ml.
WB 1/10000 - 1/50000. Detects a band of approximately 180 kDa (predicted molecular weight: 183 kDa).
IHC-P 1/100 - 1/250. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Flow Cyt Use at an assay dependent concentration.
  • Application notesIs unsuitable for IP.
  • Target

    Anti-Topoisomerase II alpha + Topoisomerase II beta antibody [EPR5377] images

    • ab109524 staining Topoisomerase II alpha + Topoisomerase II beta in the human cell line HeLa (human cervix adenocarcinoma) by flow cytometry. Cells were fixed with 4% paraformaldehyde, permiabilised with 90% methanol and the sample was incubated with the primary antibody at a dilution of 1/20. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/2000 was used as the secondary antibody.

      Isoytype control: Rabbit monoclonal IgG (Black)

      Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue)

    • ICC/IF image of ab109524 stained HeLa cells. The cells were 4% Formaldehyde 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 (ab109524, 10µg/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.

    • All lanes : Anti-Topoisomerase II alpha + Topoisomerase II beta antibody [EPR5377] (ab109524) at 1/1000 dilution

      Lane 1 : ab109524 on TOP II alpha recombinant protein 10ng
      Lane 2 : Anti-His rabbit serum on TOP II alpha recombinant protein 10ng

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

      Predicted band size : 183 kDa
      Observed band size : 24 kDa (why is the actual band size different from the predicted?)


      Exposure time : 1 second

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

      TOP II alpha recombinant protein (aa 1-220) containing a his-tag.

    • ab109524, at a 1/100 dilution, staining Human Topoisomerase II beta in breast tissue, using Immunohistochemistry, Formalin/PFA-fixed paraffin-embedded tissue.
    • All lanes : Anti-Topoisomerase II alpha + Topoisomerase II beta antibody [EPR5377] (ab109524) at 1/50000 dilution

      Lane 1 : Jurkat cell lysate
      Lane 2 : K562 cell lysate
      Lane 3 : Raw264.7 cell lysate
      Lane 4 : PC-12 cell lysate

      Lysates/proteins at 10 µg per lane.


      Predicted band size : 183 kDa
      Observed band size : 180 kDa (why is the actual band size different from the predicted?)

    References for Anti-Topoisomerase II alpha + Topoisomerase II beta antibody [EPR5377] (ab109524)

    This product has been referenced in:
    • Kouzine F  et al. Transcription-dependent dynamic supercoiling is a short-range genomic force. Nat Struct Mol Biol 20:396-403 (2013). Human . Read more (PubMed: 23416947) »

    See 1 Publication for this product

    Product Wall

    Application Western blot
    Loading amount 10 µg
    Gel Running Conditions Reduced Denaturing (4-12 Bis tris)
    Sample Human Cell lysate - whole cell (293T)
    Specification 293T
    Blocking step Milk as blocking agent for 12 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
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    Submitted Jun 20 2014

    Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"