Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)

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Western blot - Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)
  • Immunoprecipitation - Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)
  • Immunocytochemistry/ Immunofluorescence - Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)
  • Flow Cytometry - Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)
  • Immunoprecipitation - Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)
  • Western blot - Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)
  • Western blot - Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)
  • Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR13809(B)] to USP9x - N-terminal
  • Suitable for: IHC-P, WB, ICC/IF, IP, Flow Cyt
  • Knockout validated
  • Reacts with: Mouse, Human

Overview

  • Product name

    Anti-USP9x antibody [EPR13809(B)] - N-terminal
    See all USP9x primary antibodies

  • Description

    Rabbit monoclonal [EPR13809(B)] to USP9x - N-terminal

  • Host species

    Rabbit

  • Tested Applications & Species

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

  • Immunogen

    Synthetic peptide within Human USP9x aa 1-100 (Cysteine residue). The exact sequence is proprietary.
    Database link: Q93008

  • Positive control

    • WB: HeLa, Hap1, T84, NIH3T3, Jurkat and HepG2 cell lysates. IHC-P: Human kidney and testis tissues. IP: HeLa and NIH3T3 cell lysates. Flow Cyt: Jurkat cells. ICC/IF: HeLa cells.

  • 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.

    The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing the problem with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation.

    One factor contributing to the crisis is the use of antibodies that are not suitable. This can lead to misleading results and the use of incorrect data informing project assumptions and direction. To help address this challenge, we have introduced an application and species grid on our primary antibody datasheets to make it easy to simplify identification of the right antibody for your needs.

    Learn more here.

Properties

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab180191 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Guaranteed

Tested applications are guaranteed to work and covered by our Abpromise guarantee.

Predicted

Predicted to work for this combination of applications and species but not guaranteed.

Incompatible

Does not work for this combination of applications and species.

Application Species
Flow Cyt
Human
ICC/IF
Human
IHC-P
Human
IP
Mouse
Human
WB
Mouse
Human
All applications
Rat
Application Abreviews Notes
IHC-P
1/50 - 1/100. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Antigen retrieval is recommended.
WB (1)
1/1000 - 1/10000. Predicted molecular weight: 292 kDa.
ICC/IF
1/100 - 1/250.
IP
1/20.
Flow Cyt
1/10 - 1/100.

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

 
Notes
IHC-P
1/50 - 1/100. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Antigen retrieval is recommended.
WB
1/1000 - 1/10000. Predicted molecular weight: 292 kDa.
ICC/IF
1/100 - 1/250.
IP
1/20.
Flow Cyt
1/10 - 1/100.

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

 

Target

  • Function

    Deubiquitinase involved both in the processing of ubiquitin precursors and of ubiquitinated proteins. May therefore play an important role regulatory role at the level of protein turnover by preventing degradation of proteins through the removal of conjugated ubiquitin. Essential component of TGF-beta/BMP signaling cascade. Regulates chromosome alignment and segregation in mitosis by regulating the localization of BIRC5/survivin to mitotic centromeres. Specifically hydrolyzes both 'Lys-29'- and 'Lys-33'-linked polyubiquitins chains. Specifically deubiquitinates monoubiquitinated SMAD4, opposing the activity of E3 ubiquitin-protein ligase TRIM33.

  • Tissue specificity

    Widely expressed in embryonic and adult tissues.

  • Sequence similarities

    Belongs to the peptidase C19 family.

  • Cellular localization

    Cytoplasm.
  • Information by UniProt

  • Database links

    • Alternative names

      • Deubiquitinating enzyme FAF X antibody
      • Deubiquitinating enzyme FAF-X antibody
      • DFFRX antibody
      • Drosophila fat facets related X linked antibody
      • FAF antibody
      • Fafl antibody
      • Fam antibody
      • Fat facets homolog antibody
      • Fat facets in mammals antibody
      • Fat facets protein related X linked antibody
      • Fat facets protein related, X-linked antibody
      • Fat facets protein-related antibody
      • hFAM antibody
      • MRX99 antibody
      • Probable ubiquitin carboxyl terminal hydrolase FAF X antibody
      • Probable ubiquitin carboxyl-terminal hydrolase FAF-X antibody
      • Ubiquitin carboxyl-terminal hydrolase FAM antibody
      • Ubiquitin specific peptidase 9 X linked antibody
      • Ubiquitin specific peptidase 9, X-linked antibody
      • Ubiquitin specific processing protease FAF X antibody
      • Ubiquitin specific protease 9 X chromosome antibody
      • Ubiquitin thioesterase FAF X antibody
      • Ubiquitin thiolesterase FAF X antibody
      • Ubiquitin thiolesterase FAF-X antibody
      • Ubiquitin-specific protease 9 antibody
      • Ubiquitin-specific-processing protease FAF-X antibody
      • USP9 (gene name) antibody
      • Usp9x antibody
      • USP9X_HUMAN antibody
      • Uubiquitin specific protease 9, X chromosome (fat facets like Drosophila) antibody
      • X chromosome antibody
      • X-linked antibody
      see all

    Images

    • Western blot - Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)
      Western blot - Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)
      All lanes : Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191) at 1/1000 dilution

      Lane 1 : Wild-type HeLa cell lysate
      Lane 2 : USP9X knockout HeLa cell lysate

      Lysates/proteins at 20 µg per lane.

      Performed under reducing conditions.

      Predicted band size: 292 kDa
      Observed band size: 290 kDa why is the actual band size different from the predicted?



      Lanes 1- 2: Merged signal (red and green). Green - ab180191 observed at 290 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

       ab180191 was shown to react with USP9x in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265665 (knockout cell lysate ab257790) was used. Wild-type HeLa and USP9X 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. ab180191 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) 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.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)

      Immunohistochemical analysis of paraffin embedded Human kidney tissue labeling USP9x with ab180191 at 1/50.

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

    • Immunoprecipitation - Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)
      Immunoprecipitation - Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)

      ab180191 (purified) at 1/20 dilution immunoprecipitating ETS2 in HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 µg.
      Lane 1 (input): HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 µg
      Lane 2 (+): ab180191 & HeLa whole cell lysate
      Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab180191 in HeLa whole cell lysate

      For western blotting, ab180191 at 1/500 dilution (0.23 µg/mL) and veriBlot for IP secondary antibody (HRP) (ab131366) at 1/1000 dilution was used.

      Blocking and diluting buffer: 5% NFDM /TBST.

    • Immunocytochemistry/ Immunofluorescence - Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)
      Immunocytochemistry/ Immunofluorescence - Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)

      Immunofluorescent analysis of HeLa cells labeling USP9x with ab180191 at 1/100 (green) and DAPI staining (blue).

    • Flow Cytometry - Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)
      Flow Cytometry - Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)

      Flow Cytometrical analysis of permeabilized Jurkat cells labeling USP9x with ab180191 at 1/10 (red) or a rabbit IgG negative (green).

    • Immunoprecipitation - Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)
      Immunoprecipitation - Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)

      ab180191 (purified) at 1/20 dilution immunoprecipitating ETS2 in NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate 10 µg.

      Lane 1 (input): NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate 10 µg.
      Lane 2 (+): ab180191 & NIH/3T3 whole cell lysate
      Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab180191 in NIH/3T3 whole cell lysate

      For western blotting, ab180191 at 1/500 dilution (0.23 µg/mL) and veriBlot for IP secondary antibody (HRP) (ab131366) at 1/1000 dilution was used.

      Blocking and diluting buffer: 5% NFDM /TBST.

    • Western blot - Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)
      Western blot - Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)

      Lane 1: Wild-type HAP1 cell lysate (20 µg)
      Lane 2: USP9x knockout HAP1 cell lysate (20 µg)
      Lane 3: T84 cell lysate (20 µg)
      Lane 4: NIH3T3 cell lysate (20 µg)
      Lanes 1 - 4: Merged signal (red and green). Green - ab180191 observed at 270 kDa. Red - loading control, ab18058, observed at 117 kDa.

      ab180191 was shown to specifically react with USP9x when USP9x knockout samples were used. Wild-type and USP9x knockout samples were subjected to SDS-PAGE. ab180191 and ab18058 (loading control to Vinculin) were diluted at 1 µg/ml and 1/10 000 respectively and incubated overnight at 4°C. 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-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)
      Western blot - Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)
      All lanes : Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191) at 1/1000 dilution

      Lane 1 : Jurkat cell lysate
      Lane 2 : HeLa cell lysate
      Lane 3 : HepG2 cell lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

      Predicted band size: 292 kDa

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)

      Immunohistochemical analysis of paraffin embedded Human testis tissue labeling USP9x with ab180191 at 1/50.

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

    • Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)
      Anti-USP9x antibody [EPR13809(B)] - N-terminal (ab180191)

    References (1)

    Publishing research using ab180191? Please let us know so that we can cite the reference in this datasheet.

    ab180191 has been referenced in 1 publication.

    • Li H  et al. VCP/p97 increases BMP signaling by accelerating ubiquitin ligase Smurf1 degradation. FASEB J 33:2928-2943 (2019). PubMed: 30335548

    Customer reviews and Q&As

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    Western blot abreview for Anti-USP9x antibody [EPR13809(B)] - N-terminal

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    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HAP1 cell line)
    Gel Running Conditions
    Non-reduced Denaturing (4-12 % Bis-Tris gel)
    Loading amount
    50 µg
    Specification
    HAP1 cell line
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
    Read More

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    Submitted Oct 10 2016

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