Overview

  • Product name

    Anti-MAGE3 antibody [EPR19065]
    See all MAGE3 primary antibodies
  • Description

    Rabbit monoclonal [EPR19065] to MAGE3
  • Host species

    Rabbit
  • Specificity

    This antibody cross reacts with MAGEA2, MAGEA6, MAGEA8 and MAGEA12 and does not cross react with MAGEA1 and MAGEA4.
  • Tested applications

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

    Reacts with: Human
  • Immunogen

    Recombinant full length protein within Human MAGE3 aa 1 to the C-terminus. The exact sequence is proprietary.
    Database link: P43357

  • Positive control

    • WB: His- and vinc-tagged human MAGEA2 recombinant protein; His-tagged human MAGEA6 MAGEA8 and MAGEA12 recombinant proteins; A-375, RPMI-6226 and HeLa whole cell lysates. IHC-P: Human testis, colon cancer, glioma, melanoma and gastric cancer tissues. ICC/IF: A-375 and HeLa cells. IP: A-375 whole cell lysate.
  • General notes

     This product was previously labelled as MAGEA3

     

    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: 0.05% BSA, 40% Glycerol, PBS
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EPR19065
  • Isotype

    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab223162 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. Detects a band of approximately 45-50 kDa (predicted molecular weight: 35 kDa).
IP 1/30.
ICC/IF 1/100.
IHC-P 1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Target

  • Function

    Proposed to enhance ubiquitin ligase activity of RING-type zinc finger-containing E3 ubiquitin-protein ligases. May enhance ubiquitin ligase activity of TRIM28 and stimulate p53/TP53 ubiquitination by TRIM28. Proposed to act through recruitment and/or stabilization of the Ubl-conjugating enzyme (E2) at the E3:substrate complex. May play a role in embryonal development and tumor transformation or aspects of tumor progression. In vitro promotes cell viability in melanoma cell lines. Antigen recognized on a melanoma by autologous cytolytic T-lymphocytes.
  • Tissue specificity

    Expressed in many tumors of several types, such as melanoma, head and neck squamous cell carcinoma, lung carcinoma and breast carcinoma, but not in normal tissues except for testes and placenta. Never expressed in kidney tumors, Leukemias and lymphomas.
  • Sequence similarities

    Contains 1 MAGE domain.
  • Information by UniProt
  • Database links

  • Alternative names

    • Antigen MZ2 D antibody
    • Antigen MZ2-D antibody
    • Cancer/testis antigen 1.3 antibody
    • Cancer/testis antigen family 1 member 3 antibody
    • CT1.3 antibody
    • HIP8 antibody
    • HYPD antibody
    • MAGA3_HUMAN antibody
    • MAGE 3 antigen antibody
    • MAGE family member A3 antibody
    • MAGE-3 antigen antibody
    • MAGE3 antibody
    • MAGEA3 antibody
    • MAGEA6 antibody
    • Melanoma antigen family A 3 antibody
    • Melanoma associated antigen 3 antibody
    • Melanoma-associated antigen 3 antibody
    • MGC1461 antibody
    see all

Images

  • Lane 1 : Anti-MAGE3 antibody [EPR19065] (ab223162) at 1/20000 dilution
    Lanes 2 & 4 : Anti-MAGE3 antibody [EPR19065] (ab223162) at 1/10000 dilution
    Lane 3 : Anti-MAGE3 antibody [EPR19065] (ab223162) at 1/1000 dilution

    Lane 1 : His- and vinc-tagged human MAGEA2 recombinant protein (aa1-314)
    Lane 2 : His-tagged human MAGEA6 recombinant protein (aa1-314)
    Lane 3 : His-tagged human MAGEA8 recombinant protein (aa1-318)
    Lane 4 : His-tagged human MAGEA12 recombinant protein (aa1-314)

    Lysates/proteins at 0.01 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: 35 kDa
    Observed band size: 38,41,70 kDa
    why is the actual band size different from the predicted?



     

    Exposure time: Lane 1: 3 seconds; Lanes 2, 4: 1 second; Lane 3: 30 seconds.

    Blocking/Dilution buffer: 5% NFDM/TBST.

    Vinc-tag is derived from protein vinculin (P18206) and contains aa1-258 (29 kDa).

  • All lanes : Anti-MAGE3 antibody [EPR19065] (ab223162) at 1/1000 dilution

    Lane 1 : His-tagged human MAGEA1 recombinant protein (aa1-309)
    Lane 2 : His-tagged human MAGEA4 recombinant protein (aa1-317)

    Lysates/proteins at 0.01 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: 35 kDa


    Exposure time: 3 minutes


    Blocking/Dilution buffer: 5% NFDM/TBST.

  • All lanes : Anti-MAGE3 antibody [EPR19065] (ab223162) at 1/5000 dilution

    Lane 1 : A-375 (human malignant melanoma cell line) whole cell lysate
    Lane 2 : RPMI-8226 (human plasmacytoma, myeloma B lymphocyte cell line) whole cell lysate
    Lane 3 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate

    Lysates/proteins at 10 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: 35 kDa



     

    Exposure time:  Lanes 1,2: 5 seconds; Lane 3: 2 seconds.

    Blocking/Dilution buffer: 5% NFDM/TBST.

    The observed molecular weight is consistent with what has been described in the literature (PMID:7757970).

  • Immunohistochemical analysis of paraffin-embedded human testis tissue labeling MAGE3 with ab223162 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Nuclear staining on germ cells of human testis (PMID: 23390371). Counter stained with Hematoxylin.

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

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunohistochemical analysis of paraffin-embedded human colon cancer tissue labeling MAGE3 with ab223162 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Nuclear and cytoplasmic staining on human colon cancer (PMID: 23390371). Counter stained with Hematoxylin.

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

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunohistochemical analysis of paraffin-embedded human glioma tissue labeling MAGE3 with ab223162 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Nuclear staining on human glioma (PMID: 23946777). Counter stained with Hematoxylin.

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

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunohistochemical analysis of paraffin-embedded human melanoma tissue labeling MAGE3 with ab223162 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Nuclear and cytoplasmic staining on human melanoma (PMID: 26100270). Counter stained with Hematoxylin.

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

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunohistochemical analysis of paraffin-embedded human gastric cancer tissue labeling MAGE3 with ab223162 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Nuclear and cytoplasmic staining on human gastric cancer (PMID: 16158951). Counter stained with Hematoxylin.

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

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunohistochemical analysis of paraffin-embedded human stomach tissue labeling MAGE3 with ab223162 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

    Negative control: No staining on human stomach (PMID: 16158951).

    Counter stained with Hematoxylin.

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunofluorescent analysis of 100% methanol-fixed HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling MAGE3 with ab223162 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 and cytoplasmic staining on HeLa cell line. 

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

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution.

  • Immunofluorescent analysis of 100% methanol-fixed A-375 (human malignant melanoma cell line) cells labeling MAGE3 with ab223162 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 and cytoplasmic staining on A-375 cell line.

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

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution.

  • MAGE3 was immunoprecipitated from 0.35 mg of A-375 (human malignant melanoma cell line) whole cell lysate with ab223162 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab223162 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

    Lane 1: A-375 whole cell lysate 10 μg (Input).

    Lane 2: ab223162 IP in A-375 whole cell lysate.

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab223162 in A-375 whole cell lysate

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

    Exposure time: 10 seconds.

References

ab223162 has not yet been referenced specifically in any publications.

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