Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-OTUB1 antibody [EPR13028(B)] (ab175200)

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Overview

  • Product name
    Anti-OTUB1 antibody [EPR13028(B)]
    See all OTUB1 primary antibodies
  • Description
    Rabbit monoclonal [EPR13028(B)] to OTUB1
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, IPmore details
    Unsuitable for: Flow Cyt,ICC/IF or IHC-P
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human OTUB1 aa 200 to the C-terminus (Cysteine residue). The exact sequence is proprietary.
    Database link: Q96FW1

  • Positive control
    • HeLa, HepG2, HEK-293, HEK-293T and MCF7 cell lysate. Rat heart , Mouse heart.
  • General notes

    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.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab175200 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 - 1/10000. Predicted molecular weight: 31 kDa.
IP 1/10 - 1/100.
  • Application notes
    Is unsuitable for Flow Cyt,ICC/IF or IHC-P.

    • Target

    • Function
      Hydrolase that can remove conjugated ubiquitin from proteins and plays an important regulatory role at the level of protein turnover by preventing degradation. Regulator of T-cell anergy, a phenomenon that occurs when T-cells are rendered unresponsive to antigen rechallenge and no longer respond to their cognate antigen. Acts via its interaction with RNF128/GRAIL, a crucial inductor of CD4 T-cell anergy. Isoform 1 destabilizes RNF128, leading to prevent anergy. In contrast, isoform 2 stabilizes RNF128 and promotes anergy. Surprisingly, it regulates RNF128-mediated ubiquitination, but does not deubiquitinate polyubiquitinated RNF128. Deubiquitinates estrogen receptor alpha (ESR1). Mediates deubiquitination of 'Lys-48'-linked polyubiquitin chains, but not 'Lys-63'-linked polyubiquitin chains. Not able to cleave di-ubiquitin. Also capable of removing NEDD8 from NEDD8 conjugates, but with a nuch lower preference compared to 'Lys-48'-linked ubiquitin.
    • Tissue specificity
      Isoform 1 is ubiquitous. Isoform 2 is expressed only in lymphoid tissues such as tonsils, lymph nodes and spleen, as well as peripheral blood mononuclear cells.
    • Sequence similarities
      Belongs to the peptidase C65 family.
      Contains 1 OTU domain.
    • Domain
      In addition to ubiquitin-binding at the Cys-91 active site, a proximal ubiquitin-binding site is also present at Cys-23 Occupancy of the active site is needed to enable tight binding to the second site. Distinct binding sites for the ubiquitins may allow to discriminate among different isopeptide linkages (i.e. 'Lys-48'-, 'Lys-63'-linked polyubiquitin) in polyubiquitin substrates and achieve linkage-specific deubiquitination.
    • Cellular localization
      Cytoplasm.
    • Information by UniProt
    • Database links
      see all
    • Alternative names
      • Deubiquitinating enzyme OTUB1 antibody
      • hOTU1 antibody
      • HSPC263 antibody
      • OTB1 antibody
      • OTU domain containing ubiquitin aldehyde binding protein 1 antibody
      • OTU domain, ubiquitin aldehyde binding 1 antibody
      • OTU domain-containing Ubal-binding protein 1 antibody
      • OTU domain-containing ubiquitin aldehyde-binding protein 1 antibody
      • OTU-domain Ubal-binding 1 antibody
      • OTU1 antibody
      • Otub1 antibody
      • OTUB1_HUMAN antibody
      • Otubain 1 antibody
      • Otubain-1 antibody
      • Ubiquitin specific processing protease OTUB1 antibody
      • Ubiquitin thioesterase OTUB1 antibody
      • ubiquitin-specific protease otubain 1 antibody
      • Ubiquitin-specific-processing protease OTUB1 antibody
      see all

    Images

    • Western blot - Anti-OTUB1 antibody [EPR13028(B)] (ab175200)
      Western blot - Anti-OTUB1 antibody [EPR13028(B)] (ab175200)

      Lane 1: Wild type HAP1 whole cell lysate (20 µg)
      Lane 2: OTUB1 knockout HAP1 whole cell lysate (20 µg)
      Lane 3: HeLa whole cell lysate (20 µg)
      Lane 4: HEK-293 whole cell lysate (20 µg)

      Lanes 1 - 4: Merged signal (red and green). Green - ab175200 observed at 35 kDa. Red - loading control, ab18058, observed at 130 kDa.

      ab175200 was shown to specifically react with OTUB1 in wild type cells as signal was lost in OTUB1 knockout cells. Wild-type and OTUB1 knockout samples were subjected to SDS-PAGE. ab175200 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/1000 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.

    • Immunoprecipitation - Anti-OTUB1 antibody [EPR13028(B)] (ab175200)
      Immunoprecipitation - Anti-OTUB1 antibody [EPR13028(B)] (ab175200)

      ab175200 (purified) at 1:50 dilution (2ug) immunoprecipitating OTUB1 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate.
      Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10ug
      Lane 2 (+): ab175200 & HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
      Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab175200 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
      For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
      Blocking and diluting buffer: 5% NFDM/TBST.

    • Western blot - Anti-OTUB1 antibody [EPR13028(B)] (ab175200)
      Western blot - Anti-OTUB1 antibody [EPR13028(B)] (ab175200)
      All lanes : Anti-OTUB1 antibody [EPR13028(B)] (ab175200) at 1/1000 dilution (purified)

      Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
      Lane 2 : Mouse heart whole tissue lysates
      Lane 3 : Rat heart whole tissue lysates

      Lysates/proteins at 15 µg per lane.

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

      Predicted band size: 31 kDa
      Observed band size: 31 kDa



      Blocking and diluting buffer: 5% NFDM/TBST

    • Immunoprecipitation - Anti-OTUB1 antibody [EPR13028(B)] (ab175200)
      Immunoprecipitation - Anti-OTUB1 antibody [EPR13028(B)] (ab175200)

      Western blot analysis on immunoprecipitation pellet from MCF7 cell lysate labeling OTUB1 with unpurified ab175200 at 1/10 dilution.

    • Western blot - Anti-OTUB1 antibody [EPR13028(B)] (ab175200)
      Western blot - Anti-OTUB1 antibody [EPR13028(B)] (ab175200)
      All lanes : Anti-OTUB1 antibody [EPR13028(B)] (ab175200) at 1/1000 dilution (unpurified)

      Lane 1 : HeLa cell lysate
      Lane 2 : HepG2 cell lysate
      Lane 3 : 293T cell lysate
      Lane 4 : MCF7 cell lysate

      Lysates/proteins at 10 µg per lane.

      Predicted band size: 31 kDa

    References

    This product has been referenced in:
    • Pasupala N  et al. OTUB1 non-catalytically stabilizes the E2 ubiquitin-conjugating enzyme UBE2E1 by preventing its autoubiquitination. J Biol Chem 293:18285-18295 (2018). Read more (PubMed: 30282802) »
    See 1 Publication for this product

    Publishing research using ab175200? Please let us know so that we can cite the reference in this datasheet.

    Customer reviews and Q&As

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    1-3 of 3 Abreviews or Q&A

    Western blot abreview for Anti-OTUB1 antibody [EPR13028(B)]

     Inconclusive
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HAP1 cell line)
    Gel Running Conditions
    Non-reduced Denaturing (gradient 4-12%)
    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

    Abcam user community

    Verified customer

    Submitted Feb 21 2017

    Immunoprecipitation abreview for Anti-OTUB1 [EPR13028(B)] antibody

     Excellent
    Abreviews
    abreview image
    Application
    Immunoprecipitation
    Immuno-precipitation step
    Protein A
    Sample
    Human Cell lysate - whole cell (HEK293 cells)
    Specification
    HEK293 cells
    Total protein in input
    20 µg
    Read More

    Abcam user community

    Verified customer

    Submitted Nov 12 2013

    Western blot abreview for Anti-OTUB1 [EPR13028(B)] antibody

     Excellent
    Abreviews
    abreview image
    Application
    Western blot
    Loading amount
    20 µg
    Gel Running Conditions
    Reduced Denaturing
    Sample
    Human Cell lysate - whole cell (HEK293 cells)
    Specification
    HEK293 cells
    Treatment
    untreated or HA-OTUB1 transfected for 48 hours
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
    Read More

    Abcam user community

    Verified customer

    Submitted Nov 12 2013

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