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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-5T4 antibody [EPR5529] (ab134162)

Submit a review Q&A (2)References (7)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-5T4 antibody [EPR5529] (ab134162)
  • Western blot - Anti-5T4 antibody [EPR5529] (ab134162)
  • Western blot - Anti-5T4 antibody [EPR5529] (ab134162)
  • Western blot - Anti-5T4 antibody [EPR5529] (ab134162)
  • Western blot - Anti-5T4 antibody [EPR5529] (ab134162)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-5T4 antibody [EPR5529] (ab134162)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-5T4 antibody [EPR5529] (ab134162)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-5T4 antibody [EPR5529] (ab134162)
  • OI-RD Scanning - Anti-5T4 antibody [EPR5529] (ab134162)
  • Anti-5T4 antibody [EPR5529] (ab134162)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR5529] to 5T4
  • Suitable for: WB, IHC-P
  • Knockout validated
  • Reacts with: Human

Conjugates logo Related conjugates and formulations

Carrier Free

Overview

  • Product name

    Anti-5T4 antibody [EPR5529]
    See all 5T4 primary antibodies

  • Description

    Rabbit monoclonal [EPR5529] to 5T4

  • Host species

    Rabbit

  • Tested applications

    Suitable for: WB, IHC-Pmore details
    Unsuitable for: Flow Cyt,ICC/IF or IP

  • Species reactivity

    Reacts with: Human

  • Immunogen

    Synthetic peptide within Human 5T4 aa 100-200 (extracellular). The exact sequence is proprietary.
    Database link: Q13641

  • Positive control

    • WB: MCF7, HeLa, HEK-293T, HT-29, HT-1376, and Caco-2 cell lysates. IHC-P: Human placenta and endometrial cancer 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.

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

Properties

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab134162 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. Predicted molecular weight: 46 kDa.

For unpurified use at 1/500.
IHC-P
1/1000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

For unpurified use at 1/100.
Notes
WB
1/1000 - 1/10000. Predicted molecular weight: 46 kDa.

For unpurified use at 1/500.
IHC-P
1/1000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

For unpurified use at 1/100.
Application notes
Is unsuitable for Flow Cyt,ICC/IF or IP.

Target

  • Tissue specificity

    Expressed by all types of trophoblasts as early as 9 weeks of development. Specific for trophoblastic cells except for amniotic epithelium. In adult tissues, the expression is limited to a few epithelial cell types but is found on a variety of carcinoma.

  • Sequence similarities

    Contains 6 LRR (leucine-rich) repeats.
    Contains 1 LRRCT domain.
    Contains 1 LRRNT domain.

  • Cellular localization

    Membrane.
  • Information by UniProt

  • Database links

    • Form

      5T4 is a 72 kDa transmembrane glycoprotein that exhibits restricted expression in human and mouse adult tissues, is upregulated on many carcinomas and tumour expression correlates with poorer clinical outcome in ovarian, gastric and colorectal cancers. mES cells lack cell surface expression of the 5T4 antigen, and both protein and mRNA are rapidly upregulated following induction of differentiation. This proteins expression correlates with downregulation of OCT-4 and Tra-1–60 (Ward et al., 2006).

    • Alternative names

      • 5T4 antibody
      • 5T4 oncofetal antigen antibody
      • 5T4 oncofetal trophoblast glycoprotein antibody
      • 5T4 oncotrophoblast glycoprotein antibody
      • 5T4AG antibody
      • AW495680 antibody
      • M6P1 antibody
      • TPBG antibody
      • TPBG_HUMAN antibody
      • Trophoblast glycoprotein antibody
      see all

    Images

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-5T4 antibody [EPR5529] (ab134162)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-5T4 antibody [EPR5529] (ab134162)

      Immunohistochemical staining using ab134162 on paraffin embedded human placenta tissue.

    • Western blot - Anti-5T4 antibody [EPR5529] (ab134162)
      Western blot - Anti-5T4 antibody [EPR5529] (ab134162)
      All lanes : Anti-5T4 antibody [EPR5529] (ab134162) at 1/1000 dilution

      Lane 1 : Wild-type MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
      Lane 2 : TPBG knockout MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
      Lane 3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
      Lane 4 : HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate

      Lysates/proteins at 20 µg per lane.

      Performed under reducing conditions.

      Predicted band size: 46 kDa
      Observed band size: 85 kDa why is the actual band size different from the predicted?



      Lanes 1 - 4: Merged signal (red and green). Green - ab134162 observed at 85 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

      ab134162 was shown to react with 5T4 in wild-type MCF7 cells in Western blot with loss of signal observed in TPBG knockout cell line ab269499 (knockout cell lysate ab269661). Wild-type MCF7 and TPBG knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in before incubation with ab134162 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 h at room temperature before imaging.

    • Western blot - Anti-5T4 antibody [EPR5529] (ab134162)
      Western blot - Anti-5T4 antibody [EPR5529] (ab134162)
      Anti-5T4 antibody [EPR5529] (ab134162) at 1/500 dilution (unpurified) + HT-29 cell lysate at 20 µg

      Secondary
      Peroxidase conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

      Predicted band size: 46 kDa
      Observed band size: 72-80 kDa why is the actual band size different from the predicted?



      Blocking buffer and concentration: 5% NFDM/TBST.

      Diluting buffer and concentration: 5% NFDM /TBST.

    • Western blot - Anti-5T4 antibody [EPR5529] (ab134162)
      Western blot - Anti-5T4 antibody [EPR5529] (ab134162)
      Anti-5T4 antibody [EPR5529] (ab134162) at 1/5000 dilution (purified) + HT-29 cell lysate at 20 µg

      Secondary
      Peroxidase conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

      Predicted band size: 46 kDa
      Observed band size: 72-80 kDa why is the actual band size different from the predicted?



      Blocking buffer and concentration: 5% NFDM/TBST.

      Diluting buffer and concentration: 5% NFDM /TBST.

    • Western blot - Anti-5T4 antibody [EPR5529] (ab134162)
      Western blot - Anti-5T4 antibody [EPR5529] (ab134162)
      All lanes : Anti-5T4 antibody [EPR5529] (ab134162) at 1/1000 dilution (unpurified format)

      Lane 1 : HT-29 cell lysates
      Lane 2 : HT-1376 cell lysates
      Lane 3 : Caco-2 cell lysates

      Lysates/proteins at 10 µg per lane.

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

      Predicted band size: 46 kDa

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-5T4 antibody [EPR5529] (ab134162)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-5T4 antibody [EPR5529] (ab134162)

      Immunohistochemical staining using ab134162 on paraffin embedded human endometrial cancer.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-5T4 antibody [EPR5529] (ab134162)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-5T4 antibody [EPR5529] (ab134162)

      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human placenta tissue labelling 5T4 with unpurified ab134162 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-5T4 antibody [EPR5529] (ab134162)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-5T4 antibody [EPR5529] (ab134162)

      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human placenta tissue labelling 5T4 with purified ab134162 at 1/1000. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

    • OI-RD Scanning - Anti-5T4 antibody [EPR5529] (ab134162)
      OI-RD Scanning - Anti-5T4 antibody [EPR5529] (ab134162)
      Equilibrium disassociation constant (KD)
      Learn more about KD

      Click here to learn more about KD
    • Anti-5T4 antibody [EPR5529] (ab134162)
      Anti-5T4 antibody [EPR5529] (ab134162)

    References (7)

    Publishing research using ab134162? Please let us know so that we can cite the reference in this datasheet.

    ab134162 has been referenced in 7 publications.

    • Wan YL  et al. Combination Treatment with an Antibody-Drug Conjugate (A1mcMMAF) Targeting the Oncofetal Glycoprotein 5T4 and Carboplatin Improves Survival in a Xenograft Model of Ovarian Cancer. Target Oncol 14:465-477 (2019). PubMed: 31332693
    • Pirie-Shepherd SR  et al. Detecting expression of 5T4 in CTCs and tumor samples from NSCLC patients. PLoS One 12:e0179561 (2017). PubMed: 28727782
    • Abdalla Z  et al. Loss of epithelial markers is an early event in oral dysplasia and is observed within the safety margin of dysplastic and T1 OSCC biopsies. PLoS One 12:e0187449 (2017). PubMed: 29216196
    • Guo X  et al. 5T4-specific chimeric antigen receptor modification promotes the immune efficacy of cytokine-induced killer cells against nasopharyngeal carcinoma stem cell-like cells. Sci Rep 7:4859 (2017). PubMed: 28687750
    • Kerk SA  et al. 5T4-Targeted Therapy Ablates Cancer Stem Cells and Prevents Recurrence of Head and Neck Squamous Cell Carcinoma. Clin Cancer Res 23:2516-2527 (2017). PubMed: 27780858
    • Loh KH  et al. Proteomic Analysis of Unbounded Cellular Compartments: Synaptic Clefts. Cell 166:1295-1307.e21 (2016). PubMed: 27565350
    • Sapra P  et al. Long-term tumor regression induced by an antibody-drug conjugate that targets 5T4, an oncofetal antigen expressed on tumor-initiating cells. Mol Cancer Ther 12:38-47 (2013). IHC-P . PubMed: 23223830

    Customer reviews and Q&As

    Show All Reviews Q&A
    Submit a review Submit a question

    1-2 of 2 Abreviews or Q&A

    Question

    when an antibody is not recommended for IHC, have you tested that product for all applications or is it sometimes that IHC simply wasn't tested?

    if you're looking to do IHC with said antibody high pretreatment temp and pressure seem to help.

    Read More
    Answer



    When an antibody is noted as 'unsuitable', it has been tested in that application. It may be that optimized conditions or samples were not used during this QC so I am happy to see that you have had success with your IHC. Thanks very much for your protocol advice. I will pass this on!

    Read More

    Question

    Could I get the details of your pervanadate treatment in the WB image on the datasheet? The concentration, incubation time, and method of reconstituting would be very helpful.

    Read More
    Answer

    Thank you for contacting us. Please find below the notes on the image with pervanadate.


    Prepare 500mM stock Sodium Orthovanadate in PBS. To make
    100X Pervanadate right before experiment, add 400ul 500mM Sodium Orthovanadate to 1560ul H2O and then 40ul H2O2(Fisher #H325-100), vortex and incubate at RT for 5-10min.

    Cells are serum-starved for overnight, and then treated with freshly made Pervanadate for 20min at 37 degrees C.


    I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

    Read More

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