Overview

  • Product name

    Anti-RUNX3 antibody [EPR20687]
    See all RUNX3 primary antibodies
  • Description

    Rabbit monoclonal [EPR20687] to RUNX3
  • Host species

    Rabbit
  • Tested applications

    Suitable for: ChIP, WB, IP, Flow Cyt, ICC/IF, IHC-Pmore details
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Synthetic peptide within Human RUNX3 aa 150-250. The exact sequence is proprietary.
    Database link: Q13761

  • Positive control

    • WB: Raji and Ramos cell lysate; human tonsil tissue lysate. IHC: Human stomach, gastric cancer and diffuse large B cell lymphoma tissue. ICC/IF: Ramos and Raji cells. FC: Raji cells. IP: Raji cell lysate.
  • 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

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
  • Storage buffer

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

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EPR20687
  • Isotype

    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab224641 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
ChIP Use at an assay dependent concentration.
WB 1/1000. Detects a band of approximately 44, 46 kDa (predicted molecular weight: 44 kDa).
IP 1/30.
Flow Cyt 1/500.
ICC/IF 1/100.
IHC-P 1/1000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Target

  • Function

    CBF binds to the core site, 5'-PYGPYGGT-3', of a number of enhancers and promoters, including murine leukemia virus, polyomavirus enhancer, T-cell receptor enhancers, lck, IL-3 and GM-CSF promoters.
  • Sequence similarities

    Contains 1 Runt domain.
  • Domain

    A proline/serine/threonine rich region at the C-terminus is necessary for transcriptional activation of target genes.
  • Post-translational
    modifications

    Phosphorylated on tyrosine residues by SRC. Phosphorylated by LCK and FYN.
  • Cellular localization

    Nucleus. Cytoplasm. The tyrosine phosphorylated form localizes to the cytoplasm.
  • Information by UniProt
  • Database links

  • Alternative names

    • Acute myeloid leukemia 2 protein antibody
    • Acute myeloid leukemia gene 2 antibody
    • AML 2 antibody
    • AML2 antibody
    • CBF alpha 3 antibody
    • CBF-alpha-3 antibody
    • CBFA 3 antibody
    • CBFA3 antibody
    • Core binding factor alpha 3 subunit antibody
    • core binding factor antibody
    • Core binding factor runt domain alpha subunit 3 antibody
    • Core binding factor subunit alpha 3 antibody
    • core-binding factor antibody
    • Core-binding factor subunit alpha-3 antibody
    • Oncogene AML 2 antibody
    • Oncogene AML-2 antibody
    • PEA2 alpha C antibody
    • PEA2-alpha C antibody
    • PEBP2 alpha C antibody
    • PEBP2-alpha C antibody
    • Pebp2a3 antibody
    • PEBP2aC antibody
    • Polyomavirus enhancer binding protein 2 alpha C subunit antibody
    • Polyomavirus enhancer-binding protein 2 alpha C subunit antibody
    • runt domain alpha subunit 3 antibody
    • runt related transcription factor 3 antibody
    • Runt-related transcription factor 3 antibody
    • RUNX 3 antibody
    • Runx3 antibody
    • RUNX3_HUMAN antibody
    • SL3 3 enhancer factor 1 alpha C subunit antibody
    • SL3-3 enhancer factor 1 alpha C subunit antibody
    • SL3/AKV core binding factor alpha C subunit antibody
    • SL3/AKV core-binding factor alpha C subunit antibody
    • Transcription factor AML2 antibody
    see all

Images

  • All lanes : Anti-RUNX3 antibody [EPR20687] (ab224641) at 1/1000 dilution

    Lane 1 : Wild-type HAP1 whole cell lysate
    Lane 2 : RUNX3 knockout HAP1 whole cell lysate
    Lane 3 : Ramos whole cell lysate
    Lane 4 : HeLa whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 44 kDa



    Lanes 1 - 4: Merged signal (red and green). Green - ab224641 observed at 44-46 kDa. Red - loading control, ab130007, observed at 130 kDa.

    ab224641 was shown to specifically react with RUNX3 in wild-type HAP1 cells as signal was lost in RUNX3 knockout cells. Wild-type and RUNX3 knockout samples were subjected to SDS-PAGE. Ab224641 and ab130007 (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.

  • All lanes : Anti-RUNX3 antibody [EPR20687] (ab224641) at 1/1000 dilution

    Lane 1 : Raji (human Burkitt's lymphoma cell line) cell lysate
    Lane 2 : Ramos (human Burkitt's lymphoma cell line) cell lysate
    Lane 3 : MCF7 (human breast adenocarcinoma cell line) 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.

    Performed under reducing conditions.

    Predicted band size: 44 kDa
    Observed band size: 44, 46 kDa
    why is the actual band size different from the predicted?


    Exposure time: 5 seconds


    Blocking/Dilution: 5% NFDM/TBST.

    Negative control: MCF7 (PMID:21706051).

    This target detects both predicted isoforms 44KDa and 46KDa that consistent with what has been described in the literature (PMID:23700080).

  • Immunohistochemical analysis of paraffin-embedded human stomach tissue labeling RUNX3 with ab224641 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051), ready to use. Nuclear staining on lymphoid cells of human stomach (PMID:15514019; PMID:21786422) is observed. Counter stained with hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051), ready to use.

    Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Ramos (human Burkitt's lymphoma cell line) cells labeling RUNX3 with ab224641 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 on Ramos cell line. DAPI (blue) and anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) (red) at 1/200 dilution were used as counterstains.

    The negative controls are as follows:

    Negative control: MCF7 (human breast adenocarcinoma cell line) (PMID: 21706051).

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is AlexaFluor®488 Goat anti-Rabbit (ab150077) at 1/1000 dilution..

  • Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized Raji (human Burkitt's lymphoma cell line) cell line labeling RUNX3 with ab224641 at 1/500 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

  • Chromatin was prepared from U-937 (PMA treated or not) cells according to the Abcam X-ChIP protocol. Cells were fixed with 1% formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 5µg of ab224641 (red), and 20µl of protein A/G sepharose beads slurry (10µl of sepharose A beads + 10µl of sepharose G beads). 5μg of rabbit normal IgG was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

  • RUNX3 was immunoprecipitated from 0.35 mg of Raji (human Burkitt's lymphoma cell line) whole cell lysate with ab224641 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab224641 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

    Lane 1: Raji whole cell lysate 10 µg (Input). 

    Lane 2: ab224641 IP in Raji whole cell lysate (+). 

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab224641 in Raji whole cell lysate (-).

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

     

  • Anti-RUNX3 antibody [EPR20687] (ab224641) at 1/1000 dilution + Human tonsil tissue lysate at 20 µg

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

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 44 kDa


    Exposure time: 30 seconds


    Blocking/Dilution: 5% NFDM/TBST.

    This target detects both predicted isoforms 44KDa and 46KDa that consistent with what has been described in the literature (PMID:23700080).

  • Immunohistochemical analysis of paraffin-embedded human gastric cancer tissue labeling RUNX3 with ab224641 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051), ready to use. Nuclear staining on lymphoid cells of human gastric cancer (PMID:27566570) is observed. Counter stained with hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051), ready to use.

    Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Raji (human Burkitt's lymphoma cell line) cells labeling RUNX3 with ab224641 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 on Raji cell line. DAPI (blue) and anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) (red) at 1/200 dilution were used as counterstains.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is AlexaFluor®488 Goat anti-Rabbit (ab150077) at 1/1000 dilution.

  • Immunohistochemical analysis of paraffin-embedded human diffuse large B cell lymphoma tissue labeling RUNX3 with ab224641 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051), ready to use. Nuclear staining on human diffuse large B cell lymphoma (PMID:27184221) is observed. Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051), ready to use.

    Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

References

ab224641 has not yet been referenced specifically in any publications.

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