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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-OTUB1 antibody [EPR24917-75] (ab270959)

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Western blot - Anti-OTUB1 antibody [EPR24917-75] (ab270959)
  • Immunocytochemistry/ Immunofluorescence - Anti-OTUB1 antibody [EPR24917-75] (ab270959)
  • Immunocytochemistry/ Immunofluorescence - Anti-OTUB1 antibody [EPR24917-75] (ab270959)
  • Flow Cytometry (Intracellular) - Anti-OTUB1 antibody [EPR24917-75] (ab270959)
  • Flow Cytometry (Intracellular) - Anti-OTUB1 antibody [EPR24917-75] (ab270959)
  • Immunoprecipitation - Anti-OTUB1 antibody [EPR24917-75] (ab270959)
  • Western blot - Anti-OTUB1 antibody [EPR24917-75] (ab270959)
  • Western blot - Anti-OTUB1 antibody [EPR24917-75] (ab270959)
  • Anti-OTUB1 antibody [EPR24917-75] (ab270959)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR24917-75] to OTUB1
  • Suitable for: IP, ICC/IF, WB, Flow Cyt (Intra)
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Conjugates logo Related conjugates and formulations

Carrier Free

Overview

  • Product name

    Anti-OTUB1 antibody [EPR24917-75]
    See all OTUB1 primary antibodies

  • Description

    Rabbit monoclonal [EPR24917-75] to OTUB1

  • Host species

    Rabbit

  • Tested applications

    Suitable for: IP, ICC/IF, WB, Flow Cyt (Intra)more details
    Unsuitable for: IHC-P

  • Species reactivity

    Reacts with: Mouse, Rat, Human

  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: WT HEK293T, HeLa, MCF7, U-2 OS, Mouse liver, Mouse brain, Rat liver, Rat brain, C6, RAW264.7, PC-12, NIH/3T3 lysates. ICC: HeLa, WT HEK293T cells. Flow Cyt Intra: Wild-type HEK293T, HeLa cells. IP: HeLa cells.

  • General notes

    Please note: IP, FC and ICC are valid for Human samples only

    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.

Properties

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab270959 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
IP
1/30.
ICC/IF
1/250.
WB
1/1000. Predicted molecular weight: 31 kDa.
Flow Cyt (Intra)
1/500.
Notes
IP
1/30.
ICC/IF
1/250.
WB
1/1000. Predicted molecular weight: 31 kDa.
Flow Cyt (Intra)
1/500.
Application notes
Is unsuitable for 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 [EPR24917-75] (ab270959)
    Western blot - Anti-OTUB1 antibody [EPR24917-75] (ab270959)
    All lanes : Anti-OTUB1 antibody [EPR24917-75] (ab270959) at 1/1000 dilution

    Lane 1 : Wild-type HEK293T (human embryonic kidney epithelial cell) whole cell lysate
    Lane 2 : OTUB1 knockout HEK293T (ab266551) whole cell lysate
    Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (ab216773) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (ab216776) at 1/10000 dilution

    Predicted band size: 31 kDa



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

    Lanes 1-3: Merged signal (red and green). Green -ab270959 observed at 31kDa. Red - loading control ab8245 observed at 36 kDa. ab270959 Anti-OTUB1 antibody [EPR24917-75] was shown to specifically react with OTUB1 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266551 (knockout cell lysate ab257569) was used. Wild-type and OTUB1 knockout samples were subjected to SDS-PAGE.

    ab270959 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 10000 dilution for 1 hour at room temperature before imaging.

  • Immunocytochemistry/ Immunofluorescence - Anti-OTUB1 antibody [EPR24917-75] (ab270959)
    Immunocytochemistry/ Immunofluorescence - Anti-OTUB1 antibody [EPR24917-75] (ab270959)

    Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa cells labelling OTUB1 with ab270959 at 1/250 (2.204 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing nuclear and cytoplasmic staining in HeLa cell line is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

    Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution.

  • Immunocytochemistry/ Immunofluorescence - Anti-OTUB1 antibody [EPR24917-75] (ab270959)
    Immunocytochemistry/ Immunofluorescence - Anti-OTUB1 antibody [EPR24917-75] (ab270959)

    Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized OTUB1 KO HEK293T (ab266551) cells labelling OTUB1 with ab270959 at 1/250 (2.204 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing no staining in OTUB1 KO HEK293T cell line and nuclear and cytoplasmic staining in Parental HEK293T is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

    Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution.

  • Flow Cytometry (Intracellular) - Anti-OTUB1 antibody [EPR24917-75] (ab270959)
    Flow Cytometry (Intracellular) - Anti-OTUB1 antibody [EPR24917-75] (ab270959)

    Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Wild-type HEK293T (human embryonic kidney epithelial cell, Right)/ OTUB1 KO HEK293T (Left) cells labelling OTUB1 with ab270959 at 1/500 dilution (0.1ug)/ (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody. Positive staining on 293T cells (ab255449), while no staining on OTUB1 knockout HEK-293T cells (ab266551).

  • Flow Cytometry (Intracellular) - Anti-OTUB1 antibody [EPR24917-75] (ab270959)
    Flow Cytometry (Intracellular) - Anti-OTUB1 antibody [EPR24917-75] (ab270959)

    Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (Human cervix adenocarcinoma epithelial cell) cells labelling OTUB1 with ab270959 at 1/500 dilution (0.1ug)/ (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

  • Immunoprecipitation - Anti-OTUB1 antibody [EPR24917-75] (ab270959)
    Immunoprecipitation - Anti-OTUB1 antibody [EPR24917-75] (ab270959)

    OTUB1 was immunoprecipitated from 0.35 mg HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate 10ug with ab270959 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab270959 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

    Lane 1: HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate 10ug

    Lane 2: ab270959 IP in HeLa whole cell lysate

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab270959 in HeLa whole cell lysate

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

    Exposure time: 7 seconds

     

  • Western blot - Anti-OTUB1 antibody [EPR24917-75] (ab270959)
    Western blot - Anti-OTUB1 antibody [EPR24917-75] (ab270959)
    All lanes : Anti-OTUB1 antibody [EPR24917-75] (ab270959) at 1/1000 dilution

    Lane 1 : MCF7 (human breast adenocarcinoma epithelial cell), whole cell lysate
    Lane 2 : U-2 OS(human bone osteosarcoma epithelial cell), whole cell lysate
    Lane 3 : Mouse liver tissue lysate
    Lane 4 : Mouse brain tissue lysate
    Lane 5 : Rat liver tissue lysate
    Lane 6 : Rat brain tissue lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 31 kDa



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

     Exposure time: 26 seconds

     

  • Western blot - Anti-OTUB1 antibody [EPR24917-75] (ab270959)
    Western blot - Anti-OTUB1 antibody [EPR24917-75] (ab270959)
    All lanes : Anti-OTUB1 antibody [EPR24917-75] (ab270959) at 1/1000 dilution

    Lane 1 : C6 (rat glial tumor glial cell), whole cell lysate
    Lane 2 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate
    Lane 3 : PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate
    Lane 4 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 31 kDa



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

     Exposure time: 15 seconds

     

  • Anti-OTUB1 antibody [EPR24917-75] (ab270959)
    Anti-OTUB1 antibody [EPR24917-75] (ab270959)

Protocols

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

References (0)

Publishing research using ab270959? Please let us know so that we can cite the reference in this datasheet.

ab270959 has not yet been referenced specifically in any publications.

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