Recombinant
RabMAb

Recombinant Anti-Bmi1 (phospho T275) antibody [EPR19848] (ab213723)

Overview

  • Product name

    Anti-Bmi1 (phospho T275) antibody [EPR19848]
    See all Bmi1 primary antibodies
  • Description

    Rabbit monoclonal [EPR19848] to Bmi1 (phospho T275)
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, Dot blot, ICC/IF, Flow Cyt, IPmore details
  • Species reactivity

    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide within Mouse Bmi1 aa 250 to the C-terminus (phospho T275). The exact sequence is proprietary.
    Database link: P25916

  • Positive control

    • WB: HEK-293T cells transfected with DDDDK and mCherry-tagged mouse Bmi1 (+/- exposure to UV light). Dot blot: Bmi1 (phospho T275) peptide. ICC/IF: HEK-293T cells transfected with DDDDK and mCherry-tagged mouse Bmi1 (exposed to UV light) and U2OS cells. Flow cytometry: HEK-293T cells transfected with DDDDK and mCherry-tagged mouse Bmi1. IP: HEK-293T cells transfected with DDDDK and mCherry-tagged mouse Bmi1 (exposed to UV light).
  • 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.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab213723 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 1/2000. Predicted molecular weight: 37 kDa.
Dot blot 1/1000.
ICC/IF 1/100.
Flow Cyt 1/500.
IP 1/30.

Target

  • Function

    Component of the Polycomb group (PcG) multiprotein PRC1 complex, a complex required to maintain the transcriptionally repressive state of many genes, including Hox genes, throughout development. PcG PRC1 complex acts via chromatin remodeling and modification of histones; it mediates monoubiquitination of histone H2A 'Lys-119', rendering chromatin heritably changed in its expressibility. In the PRC1 complex, it is required to stimulate the E3 ubiquitin-protein ligase activity of RNF2/RING2.
  • Sequence similarities

    Contains 1 RING-type zinc finger.
  • Post-translational
    modifications

    Monoubiquitinated (By similarity). May be polyubiquitinated; which does not lead to proteasomal degradation.
  • Cellular localization

    Nucleus. Cytoplasm.
  • Information by UniProt
  • Database links

  • Alternative names

    • B lymphoma Mo MLV insertion region (mouse) antibody
    • B lymphoma Mo MLV insertion region 1 homolog antibody
    • Bmi 1 antibody
    • BMI1 antibody
    • BMI1 polycomb ring finger oncogene antibody
    • BMI1_HUMAN antibody
    • Flvi 2/bmi 1 antibody
    • FLVI2/BMI1 antibody
    • MGC12685 antibody
    • Murine leukemia viral (bmi 1) oncogene homolog antibody
    • Oncogene BMI 1 antibody
    • PCGF 4 antibody
    • PCGF4 antibody
    • Polycomb complex protein BMI 1 antibody
    • Polycomb complex protein BMI-1 antibody
    • Polycomb group protein Bmi1 antibody
    • Polycomb group ring finger 4 antibody
    • Polycomb group RING finger protein 4 antibody
    • RING finger protein 51 antibody
    • RNF 51 antibody
    • RNF51 antibody
    see all

Images

  • All lanes : Anti-Bmi1 (phospho T275) antibody [EPR19848] (ab213723) at 1/2000 dilution

    Lane 1 : HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) transfected with DDDDK and mCherry-tagged mouse Bmi1 (WT) expression vector, whole cell lysate
    Lane 2 : HEK-293T transfected with DDDDK and mCherry-tagged mouse Bmi1 T275A mutant expression vector, whole cell lysate
    Lane 3 : HEK-293T transfected with DDDDK and mCherry-tagged mouse Bmi1 (WT) expression vector, followed by treatment with 800 J/m² UV, then cultured in DMEM containing 10% FBS for 30 minutes, whole cell lysate
    Lane 4 : HEK-293T transfected with DDDDK and mCherry-tagged mouse Bmi1 T275A mutant expression vector, followed by treatment with 800 J/m² UV, then cultured in DMEM containing 10% FBS for 30 minutes, 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

    Developed using the ECL technique.

    Predicted band size: 37 kDa
    Observed band size: 43 kDa
    why is the actual band size different from the predicted?


    Exposure time: 3 seconds


    Blocking and dilution buffer: 2% BSA/TBST

    The phosphorylation of Bmi1 at T275 is increased by UV treatment.

    The expression plasmids were kindly provided by Dr. Wei Guo from Tsinghua University.

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293T cells (human epithelial cell line from embryonic kidney transformed with large T antigen) transfected with DDDDK and mCherry-tagged mouse Bmi1 (WT) expression vector labeling Bmi1 (phospho T275) with ab213723 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining in HEK-293T cells transfected with DDDDK and mCherry-tagged mouse Bmi1 (WT) expression vector. No staining was observed in HEK-293T cells transfected with DDDDK and mCherry-tagged mouse Bmi1 T275A mutant expression vector.

    The nuclear counterstain is DAPI (blue). Tubulin is detected with Anti-Tubulin antibody [YOL1/34] - Microtubule Marker (Alexa Fluor® 647) (ab195884) at 1/200 dilution (white). 

    The negative controls are as follows:
    -ve control 1: PBS instead of primary antibody (HEK-293T transfected with wild-type BMi1), followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution.
    -ve control 2: PBS instead of primary antibody (HEK-293T transfected with Bmi1 T275A construct), followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution.

    Plasmids were kindly provided by Dr. Wei Guo from Tsinghua University.

  • Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cell line transfected with DDDDK and mCherry-tagged mouse Bmi1 T275A mutant expression vector (left), or DDDDK and mCherry-tagged mouse Bmi1 WT expression vector (right) labeling Bmi1 (phospho T275) with ab213723 at 1/500 (red) compared with a Rabbit monoclonal IgG (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (black). 

    Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077), at 1/2000 dilution was used as the secondary antibody.

    The cells were gated on the mCherry positive population.

    The plasmids were kindly provided by Dr. Wei Guo from Tsinghua University.

  • Bmi1 (phospho T275) was immunoprecipitated from 0.35 mg of HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) (transfected with DDDDK and mCherry-tagged mouse Bmi1 (WT) expression vector, followed by treatment with 800 J/m² UV, then cultured in DMEM containing 10% FBS for 30 minutes) whole cell lysate with ab213723 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab213723 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

    Lane 1: HEK-293T (transfected / UV treated) whole cell lysate 10 μg (Input).

    Lane 2: ab213723 IP in HEK-293T (transfected / UV treated) lysate.

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab213723 in HEK-293T (transfected / UV treated) whole cell lysate.

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

    Exposure time: 10 seconds.

  • Dot blot analysis of Bmi1 (phospho T275) labeled with ab213723 at 1/1,000 dilution.

    Lane 1: Bmi1 (phospho T275) peptide.

    Lane 2: Bmi1 non-phospho peptide.

    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100,000 dilution was used as secondary antibody.

    Blocking and dilution buffer: 5% NFDM/TBST.

    Exposure time: 3 minutes.

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized U-2 OS (human bone osteosarcoma epithelial cell line) cells labeling Bmi1 (phospho T275) with ab213723 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing increased nuclear foci staining observed in UV-treated U-2 OS cells. The cells were treated with 50 J/m² UV, then cultured in McCoy's 5a media supplemented with 10% FBS for 2 hours.

    The nuclear counterstain is DAPI (blue). Tubulin is detected with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594)  at 1/200 dilution (red).

    The negative controls are as follows:
    -ve control 1: PBS instead of primary antibody (non-treated cells), followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution.
    -ve control 2: PBS instead of primary antibody (UV treated cells), followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution.

     

References

ab213723 has not yet been referenced specifically in any publications.

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