Astana Biomed Group, an authorized Abcam distributor in Central Asia

Anti-Rb (phospho T373) antibody [EP821Y] (ab52975)

Key features and details

  • Rabbit monoclonal [EP821Y] to Rb (phospho T373)
  • Suitable for: WB, IP, IHC-P, ICC/IF, Dot blot
  • Reacts with: Human
  • Isotype: IgG

Overview

  • Product name

    Anti-Rb (phospho T373) antibody [EP821Y]
    See all Rb primary antibodies

  • Description

    Rabbit monoclonal [EP821Y] to Rb (phospho T373)

  • Host species

    Rabbit

  • Specificity

    ab52975 only detects Rb phosphorylated on Threonine 373.

  • Tested Applications & Species

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

  • Immunogen

    A synthetic phospho-peptide corresponding to residues surrounding Thr373 of human Rb was used as immunogen. The antibody only detects Rb phosphorylated on Threonine 373.

  • Positive control

    • Jurkat nuclear cell lysate Human kidney carcinoma tissue

  • General notes

    Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

     

    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

Images

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rb (phospho T373) antibody [EP821Y] (ab52975)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rb (phospho T373) antibody [EP821Y] (ab52975)

    ab52975, at a 1/250 dilution, staining Human Rb (Phosphorylated) in Kidney carcinoma, using Immunohistochemistry, Paraffin Embedded tissue.

    Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

  • Western blot - Anti-Rb (phospho T373) antibody [EP821Y] (ab52975)
    Western blot - Anti-Rb (phospho T373) antibody [EP821Y] (ab52975)
    All lanes : Anti-Rb (phospho T373) antibody [EP821Y] (ab52975) at 1/10000 dilution

    Lane 1 : Jurkat (Human T cell leukemia T lymphocyte) serum starved for 24 h. Then 10% FBS incubated for 20 h. Whole cell lysates.
    Lane 2 : Jurkat (Human T cell leukemia T lymphocyte) serum starved for 24 h. Then 10% FBS incubated for 20 h.

    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: 106 kDa
    Additional bands at: 110 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 1 minute


    Blocking and diluting buffer: 5% NFDM/TBST

    The different result is due to the lysates preparation method. The lysate in lane 1 is prepared by 1%SDS Hot Lysate buffer method. The lysate in lane 2 is prepared by RIPA lysis method. This antibody works in 1%SDS Hot Lysates in WB.

    For Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).
  • Immunocytochemistry/ Immunofluorescence - Anti-Rb (phospho T373) antibody [EP821Y] (ab52975)
    Immunocytochemistry/ Immunofluorescence - Anti-Rb (phospho T373) antibody [EP821Y] (ab52975)

    Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% tritonX-100 permeabilized MCF7(Human breast denocarcinoma cell line) cells, Serum starved and non-starved, labeling Rb (phospho T373) with Ab52975 at 1/100 dilution followed by Goat anti-Rabbit secondary IgG AlexaFluor®488 (ab150077) secondary antibody at 1/1000 dilution (green).

    Confocal image showing nuclear staining on MCF7 cells.The number of positive cells increased after treatment with FBS (fetal bovine serum) for 48 hours, then decreased after Lambda Protein Phosphatase treatment (31℃ for 2hours ).

  • Immunoprecipitation - Anti-Rb (phospho T373) antibody [EP821Y] (ab52975)
    Immunoprecipitation - Anti-Rb (phospho T373) antibody [EP821Y] (ab52975)
    All lanes : Anti-Rb (phospho T373) antibody [EP821Y] (ab52975) at 1/500 dilution

    Lane 1 : Jurkat (human acute T cell leukemia) starved 24h then 10% FBS incubated for 20h whole cell lysate.
    Lane 2 : Jurkat (human acute T cell leukemia ) starved 24h then 10% FBS incubated for 20h whole cell lysate.
    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab52975 in Jurkat (human acute T cell leukemia ) starved 24h then 10% FBS incubated for 20h whole cell lysate.

    Lysates/proteins at 10 µg per lane.

    Secondary
    VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

    Predicted band size : 106 kDa
    Additional bands at : 110 kDa.

    Exposure time : 1 second

    Blocking and diluting buffer: 5% NFDM/TBST

    The IP lysates are prepared by RIPA method. As the WB image shows, this antibody works in 1%SDS Hot Lysate buffer in WB. So there is no band in input lane.
     
    For Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).


    Lanes 1-3 : Anti-Rb (phospho T373) antibody [EP821Y] (ab52975) at 1/500 dilution


    Lane 1 : Jurkat (human acute T cell leukemia) starved 24h then 10% FBS incubated for 20h whole cell lysate. at 10 µg
    Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab52975 in Jurkat (human acute T cell leukemia ) starved 24h then 10% FBS incubated for 20h whole cell lysate. at 10 µg

    Secondary
    Lanes 1-3 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution


    Additional bands at: 110 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 1 second
  • Western blot - Anti-Rb (phospho T373) antibody [EP821Y] (ab52975)
    Western blot - Anti-Rb (phospho T373) antibody [EP821Y] (ab52975)
    All lanes : Anti-Rb (phospho T373) antibody [EP821Y] (ab52975) at 1/10000 dilution

    Lane 1 : K562 (Human chronic myelogenous leukemia cell line from bone marrow) 2 day serum starved whole cell lysate.
    Lane 2 : K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate.
    Lane 3 : K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate. Membrane incubated with phosphatase.

    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: 106 kDa
    Observed band size: 110 kDa
    why is the actual band size different from the predicted?


    Exposure time: 30 seconds


    The lysate in this image is prepared by 1%SDS Hot Lysate buffer.

    For Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).
  • Dot Blot - Anti-Rb (phospho T373) antibody [EP821Y] (ab52975)
    Dot Blot - Anti-Rb (phospho T373) antibody [EP821Y] (ab52975)

    Dot Blot analysis of Lane 1: Rb (pT373) phospho peptide and Lane 2: Rb non-phospho peptide labeling Rb (phospho T373) with ab52975 at 1/1000 dilution. 5% NFDM/TBST was used as the diluting and blocking buffer. ab97051 Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated was used as the secondary antibody at 1/100000 dilution. Exposure time: 3 minutes.

     

     

     

     

     

     

  • Western blot - Anti-Rb (phospho T373) antibody [EP821Y] (ab52975)
    Western blot - Anti-Rb (phospho T373) antibody [EP821Y] (ab52975)
    All lanes : Anti-Rb (phospho T373) antibody [EP821Y] (ab52975) at 1/50000 dilution

    Lane 1 : Jurkat cell lysate (untreated)
    Lane 2 : Jurkat cell lysate (treated with FBS)

    Lysates/proteins at 10 µg per lane.

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

    Predicted band size: 106 kDa
    Observed band size: 110 kDa why is the actual band size different from the predicted?



    The lysate in this image is prepared by 1%SDS Hot Lysate buffer.

    For Lysate preparation protocol, please refer to the protocol book in the protocol section and/or here (downloadable copy).

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