Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574)

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Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR5270] to nmt55 / p54nrb
  • Suitable for: WB, ICC/IF, IHC-P
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Overview

  • Product name

    Anti-nmt55 / p54nrb antibody [EPR5270]
    See all nmt55 / p54nrb primary antibodies

  • Description

    Rabbit monoclonal [EPR5270] to nmt55 / p54nrb

  • Host species

    Rabbit

  • Tested applications

    Suitable for: WB, ICC/IF, IHC-Pmore details

  • Species reactivity

    Reacts with: Mouse, Rat, Human

  • Immunogen

    Synthetic peptide within Human nmt55/ p54nrb (C terminal). The exact sequence is proprietary.
    Database link: Q15233

  • Positive control

    • WB: HEK293T, PC3, MOLT4, HeLa, MCF7, A673, RAW 264.7, PC12 and NIH/3T3 cell lysates. IHC-P: Human kidney and bladder carcinoma tissues.

  • 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.

    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.

Properties

  • Form

    Liquid

  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Stable for 12 months at -20°C.

  • Dissociation constant (KD)

    KD = 2.20 x 10 -12 M
    Learn more about KD

  • Storage buffer

    pH: 7.40
    Preservative: 0.01% Sodium azide
    Constituents: 59% PBS, 0.05% BSA, 40% Glycerol
  • Concentration information loading...

  • Purity

    Protein A purified

  • Clonality

    Monoclonal

  • Clone number

    EPR5270

  • Isotype

    IgG

  • Research areas

Applications

Our Abpromise guarantee covers the use of ab133574 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. Detects a band of approximately 54 kDa (predicted molecular weight: 54 kDa).
ICC/IF 1/1500.

For unpurified use at 1/250 - 1/500.
IHC-P 1/250 - 1/500. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

Use a HRP/AP polymerized secondary antibody.

Target

  • Function

    DNA- and RNA binding protein, involved in several nuclear processes. Binds the conventional octamer sequence in double stranded DNA. Also binds single-stranded DNA and RNA at a site independent of the duplex site (By similarity). Involved in pre-mRNA splicing, probably as an heterodimer with SFPQ. Interacts with U5 snRNA, probably by binding to a purine-rich sequence located on the 3' side of U5 snRNA stem 1b. The SFPQ-NONO heteromer associated with MATR3 may play a role in nuclear retention of defective RNAs. The SFPQ-NONO heteromer may be involved in DNA unwinding by modulating the function of topoisomerase I/TOP1. The SFPQ-NONO heteromer may be involved in DNA nonhomologous end joining (NHEJ) required for double-strand break repair and V(D)J recombination and may stabilize paired DNA ends. In vitro, the complex strongly stimulates DNA end joining, binds directly to the DNA substrates and cooperates with the Ku70/G22P1-Ku80/XRCC5 (Ku) dimer to establish a functional preligation complex. Nono is involved in transcriptional regulation. The SFPQ-NONO-NR5A1 complex binds to the CYP17 promoter and regulates basal and cAMP-dependent transcriptional avtivity. NONO binds to an enhancer element in long terminal repeats of endogenous intracisternal A particles (IAPs) and activates transcription.

  • Tissue specificity

    Heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. Also found in a number of breast tumor cell lines.

  • Involvement in disease

    Note=A chromosomal aberration involving NONO may be a cause of papillary renal cell carcinoma (PRCC). Translocation t(X;X)(p11.2;q13.1) with TFE3.

  • Sequence similarities

    Contains 2 RRM (RNA recognition motif) domains.

  • Post-translational
    modifications

    The N-terminus is blocked.

  • Cellular localization

    Nucleus.
  • Information by UniProt

  • Database links

    see all

  • Alternative names

    • 52 kDa subunit antibody
    • 54 kDa nuclear RNA and DNA binding protein antibody
    • 54 kDa nuclear RNA- and DNA-binding protein antibody
    • 55 kDa nuclear protein antibody
    • DNA binding p52/p100 complex 52 kDa subunit antibody
    • DNA-binding p52/p100 complex antibody
    • NMT 55 antibody
    • NMT55 antibody
    • Non Pou domain containing octamer (ATGCAAAT) binding protein antibody
    • Non POU domain containing octamer binding antibody
    • Non POU domain containing octamer binding protein antibody
    • Non-POU domain-containing octamer-binding protein antibody
    • Nono antibody
    • NonO protein antibody
    • NONO_HUMAN antibody
    • NRB 54 antibody
    • NRB antibody
    • NRB54 antibody
    • Nuclear RNA binding protein 54kD antibody
    • P54 antibody
    • p54(nrb) antibody
    • p54nrb antibody
    • PPP1R114 antibody
    • Protein phosphatase 1 regulatory subunit 114 antibody
    see all

Images

  • Western blot - Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574)
    Western blot - Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574)
    All lanes : Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574) at 1/1000 dilution

    Lane 1 : Wild-type HEK293T cell lysate
    Lane 2 : NONO knockout HEK293T cell lysate
    Lane 3 : MOLT-4 cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 54 kDa
    Observed band size: 63 kDa
    why is the actual band size different from the predicted?



    Lanes 1-3: Merged signal (red and green). Green - ab133574 observed at 63 kDa. Red - loading control, ab8245 observed at 37 kDa.

    ab133574 Anti-nmt55 / p54nrb antibody [EPR5270] was shown to specifically react with nmt55 / p54nrb in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266244 (knockout cell lysate ab257160) was used. Wild-type and nmt55 / p54nrb knockout samples were subjected to SDS-PAGE. ab133574 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.

     

  • Western blot - Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574)
    Western blot - Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574)
    Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574) at 1/10000 dilution (purified) + PC-12 cell lysate at 20 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

    Predicted band size: 54 kDa



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

  • Western blot - Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574)
    Western blot - Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574)
    Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574) at 1/10000 dilution (purified) + RAW264.7 cell lysate at 20 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

    Predicted band size: 54 kDa



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

  • Western blot - Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574)
    Western blot - Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574)
    All lanes : Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574) at 1/10000 dilution (purified)

    Lane 1 : Molt-4 cell lysate
    Lane 2 : A673 cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

    Predicted band size: 54 kDa



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

  • Western blot - Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574)
    Western blot - Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574)
    All lanes : Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574) at 1/1000 dilution (unpurified)

    Lane 1 : PC3 cell lysate
    Lane 2 : MOLT4 cell lysate
    Lane 3 : HeLa cell lysate
    Lane 4 : MCF7 cell lysate
    Lane 5 : A673 cell lysate
    Lane 6 : RAW 264.7 cell lysate
    Lane 7 : PC12 cell lysate
    Lane 8 : NIH/3T3 cell lysate

    Lysates/proteins at 10 µg per lane.

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

    Predicted band size: 54 kDa
    Observed band size: 54 kDa

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat kidney tissue labelling nmt55/p54nrb with purified ab133574 at 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a goat anti-rabbit IgG H&L (HRP) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse liver tissue labelling nmt55/p54nrb with purified ab133574 at 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a goat anti-rabbit IgG H&L (HRP) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human prostate tissue labelling nmt55/p54nrb with purified ab133574 at 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a goat anti-rabbit IgG H&L (HRP) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574)

    Immunohistochemical analysis of formalin-fixed, paraffin-embedded Human bladder carcinoma tissue labelling nmt55 / p54nrb using unpurified ab133574 at a 1/250 dilution.

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

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574)

    Immunohistochemical analysis of formalin-fixed, paraffin-embedded Human kidney tissue labelling nmt55 /p54nrb using unpurified ab133574 at a 1/250 dilution.

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

  • Immunocytochemistry/ Immunofluorescence - Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574)
    Immunocytochemistry/ Immunofluorescence - Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574)

    Immunocytochemistry/Immunofluorescence analysis of Molt-4 (human acute lymphoblastic leukemia) cells labelling nmt55/p54nrb with purified ab133574 at 1/1500. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

    Control 1: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/500).

    Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500).

  • Other - Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574)
    Other - Anti-nmt55 / p54nrb antibody [EPR5270] (ab133574)
    Equilibrium disassociation constant (KD)
    Learn more about KD

    Click here to learn more about KD

References

Publishing research using ab133574? Please let us know so that we can cite the reference in this datasheet.

ab133574 has been referenced in 3 publications.

  • Shen W  et al. Acute hepatotoxicity of 2' fluoro-modified 5-10-5 gapmer phosphorothioate oligonucleotides in mice correlates with intracellular protein binding and the loss of DBHS proteins. Nucleic Acids Res 46:2204-2217 (2018). PubMed: 29390093
  • Hupalowska A  et al. CARM1 and Paraspeckles Regulate Pre-implantation Mouse Embryo Development. Cell 175:1902-1916.e13 (2018). PubMed: 30550788
  • Shadle SC  et al. DUX4-induced dsRNA and MYC mRNA stabilization activate apoptotic pathways in human cell models of facioscapulohumeral dystrophy. PLoS Genet 13:e1006658 (2017). ICC/IF ; Human . PubMed: 28273136

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