Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-NCE2/UBE2F antibody [EPR12932] (ab185234)

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Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR12932] to NCE2/UBE2F
  • Suitable for: WB, IHC-P, Flow Cyt, ICC/IF
  • Knockout validated
  • Reacts with: Human

Overview

  • Product name

    Anti-NCE2/UBE2F antibody [EPR12932]
    See all NCE2/UBE2F primary antibodies

  • Description

    Rabbit monoclonal [EPR12932] to NCE2/UBE2F

  • Host species

    Rabbit

  • Tested applications

    Suitable for: WB, IHC-P, Flow Cyt, ICC/IFmore details

  • Species reactivity

    Reacts with: Human

  • Immunogen

    Recombinant fragment within Human NCE2/UBE2F aa 1-150 (N terminal). The exact sequence is proprietary.
    Database link: Q969M7

  • Positive control

    • WB: HeLa, Jurkat, A375 and NTERA2/D1 cell lysates. Flow Cyt: A375 cells. ICC/IF: A375 cells. IHC-P: Human brain and Human renal carcinoma tissues.

  • General notes

     

     

     This product was previously labelled as NCE2

     

    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.

    Reproducibility is key to advancing scientific discovery and accelerating scientists’ next breakthrough.

    Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility.

    We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee.

    In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.

    We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.

    Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.

    Please check that this product meets your needs before purchasing. If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, as well as customer reviews and Q&As.

Properties

Applications

Our Abpromise guarantee covers the use of ab185234 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 21 kDa (predicted molecular weight: 21 kDa).
IHC-P 1/50 - 1/100. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Flow Cyt 1/160.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

 
ICC/IF 1/250.

Target

  • Function

    Accepts the ubiquitin-like protein NEDD8 from the UBA3-NAE1 E1 complex and catalyzes its covalent attachment to other proteins. The specific interaction with the E3 ubiquitin ligase RBX2, but not RBX1, suggests that the RBX2-UBE2F complex neddylates specific target proteins, such as CUL5.

  • Tissue specificity

    Widely expressed (at protein level).

  • Pathway

    Protein modification; protein neddylation.

  • Sequence similarities

    Belongs to the ubiquitin-conjugating enzyme family. UBE2F subfamily.
  • Information by UniProt

  • Database links

    • Alternative names

      • NCE2 antibody
      • NEDD8 carrier protein UBE2F antibody
      • NEDD8 conjugating enzyme antibody
      • NEDD8 conjugating enzyme UBE2F antibody
      • NEDD8 protein ligase UBE2F antibody
      • NEDD8-conjugating enzyme 2 antibody
      • NEDD8-conjugating enzyme UBE2F antibody
      • ube2f antibody
      • UBE2F_HUMAN antibody
      • Ubiquitin conjugating enzyme E2F antibody
      • Ubiquitin conjugating enzyme E2F (putative) antibody
      • Ubiquitin-conjugating enzyme E2 F antibody
      see all

    Images

    • Western blot - Anti-NCE2/UBE2F antibody [EPR12932] (ab185234)
      Western blot - Anti-NCE2/UBE2F antibody [EPR12932] (ab185234)
      All lanes : Anti-NCE2/UBE2F antibody [EPR12932] (ab185234) at 1/1000 dilution

      Lane 1 : Wild-type HeLa cell lysate
      Lane 2 : UBE2F knockout HeLa cell lysate
      Lane 3 : Jurkat 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: 21 kDa
      Observed band size: 24 kDa
      why is the actual band size different from the predicted?



      Lanes 1-3: Merged signal (red and green). Green - ab185234 observed at 24 kDa. Red - loading control ab8245 observed at 36 kDa. 

       ab185234 Anti-NCE2/UBE2F antibody [EPR12932] was shown to specifically react with NCE2/UBE2F in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265339 (knockout cell lysate ab257777) was used.  Wild-type and NCE2/UBE2F knockout samples were subjected to SDS-PAGE.  ab185234 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.

    • Flow Cytometry - Anti-NCE2/UBE2F antibody [EPR12932] (ab185234)
      Flow Cytometry - Anti-NCE2/UBE2F antibody [EPR12932] (ab185234)

      Flow cytometric analysis of A375 cells (2% paraformaldehyde-fixed) labeling NCE2/UBE2F with ab185234 at 1/160 dilution (red) or a rabbit IgG (negative) (green), followed by Goat anti rabbit IgG (FITC) secondary at 1/150 dilution.

    • Immunocytochemistry/ Immunofluorescence - Anti-NCE2/UBE2F antibody [EPR12932] (ab185234)
      Immunocytochemistry/ Immunofluorescence - Anti-NCE2/UBE2F antibody [EPR12932] (ab185234)

      Immunofluorescent analysis of A375 cells (4% paraformaldehyde-fixed) labeling NCE2/UBE2F with ab185234 at 1/250 dilution followed by Goat anti rabbit IgG (AlexaFluor® 555) secondary at 1/200 dilution and counter-stained with DAPI (blue).

    • Western blot - Anti-NCE2/UBE2F antibody [EPR12932] (ab185234)
      Western blot - Anti-NCE2/UBE2F antibody [EPR12932] (ab185234)
      All lanes : Anti-NCE2/UBE2F antibody [EPR12932] (ab185234) at 1/1000 dilution

      Lane 1 : Jurkat cell lysate
      Lane 2 : A375 cell lysate
      Lane 3 : NTERA2/D1 cell lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

      Predicted band size: 21 kDa

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NCE2/UBE2F antibody [EPR12932] (ab185234)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NCE2/UBE2F antibody [EPR12932] (ab185234)

      Immunohistochemical analysis of paraffin-embedded Human renal carcinoma labeling NCE2/UBE2F with ab185234 at 1/100 dilution followed by pre-diluted HRP-conjugated secondary antibody and counter-stained with Hematoxylin.

      Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NCE2/UBE2F antibody [EPR12932] (ab185234)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NCE2/UBE2F antibody [EPR12932] (ab185234)

      Immunohistochemical analysis of paraffin-embedded Human brain labeling NCE2/UBE2F with ab185234 at 1/100 dilution followed by pre-diluted HRP-conjugated secondary antibody and counter-stained with Hematoxylin.

      Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

    • Anti-NCE2/UBE2F antibody [EPR12932] (ab185234)
      Anti-NCE2/UBE2F antibody [EPR12932] (ab185234)

    References (3)

    Publishing research using ab185234? Please let us know so that we can cite the reference in this datasheet.

    ab185234 has been referenced in 3 publications.

    • Guo Z  et al. HUWE1-dependent DNA-PKcs neddylation modulates its autophosphorylation in DNA damage response. Cell Death Dis 11:400 (2020). PubMed: 32457294
    • Kabir S  et al. The CUL5 ubiquitin ligase complex mediates resistance to CDK9 and MCL1 inhibitors in lung cancer cells. Elife 8:N/A (2019). PubMed: 31294695
    • Zhou W  et al. UBE2M Is a Stress-Inducible Dual E2 for Neddylation and Ubiquitylation that Promotes Targeted Degradation of UBE2F. Mol Cell 70:1008-1024.e6 (2018). PubMed: 29932898

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