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  1. Link

    lef1-antibody-ep2030y-ab53293.pdf

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Epigenetics and Nuclear Signaling Transcription Domain Families HMG Box
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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-LEF1 antibody [EP2030Y] (ab53293)

  • Datasheet
  • SDS
Reviews (2)Q&A (3)References (13)

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Western blot - Anti-LEF1 antibody [EP2030Y] (ab53293)
  • Western blot - Anti-LEF1 antibody [EP2030Y] (ab53293)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LEF1 antibody [EP2030Y] (ab53293)
  • Immunocytochemistry/ Immunofluorescence - Anti-LEF1 antibody [EP2030Y] (ab53293)
  • Flow Cytometry (Intracellular) - Anti-LEF1 antibody [EP2030Y] (ab53293)
  • Immunocytochemistry/ Immunofluorescence - Anti-LEF1 antibody [EP2030Y] (ab53293)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LEF1 antibody [EP2030Y] (ab53293)
  • Flow Cytometry (Intracellular) - Anti-LEF1 antibody [EP2030Y] (ab53293)
  • Anti-LEF1 antibody [EP2030Y] (ab53293)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EP2030Y] to LEF1
  • Suitable for: ICC/IF, Flow Cyt (Intra), WB, IHC-P
  • Knockout validated
  • Reacts with: Human

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Overview

  • Product name

    Anti-LEF1 antibody [EP2030Y]
    See all LEF1 primary antibodies
  • Description

    Rabbit monoclonal [EP2030Y] to LEF1
  • Host species

    Rabbit
  • Tested applications

    Suitable for: ICC/IF, Flow Cyt (Intra), WB, IHC-Pmore details
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • Purchase matching WB positive control:Recombinant Human LEF1 protein
    • ICC/IF: Ramos and Jurkat cells. IHC-P: Human colon adenocarcinoma, human tonsil tissue. Flow Cyt (intra): Jurkat cells. WB: Jurkat cell lysate
  • 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

  • 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

    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EP2030Y
  • Isotype

    IgG
  • Research areas

    • Epigenetics and Nuclear Signaling
    • Transcription
    • Domain Families
    • HMG Box
    • Stem Cells
    • Lineage Markers
    • Mesoderm
    • Stem Cells
    • Signaling Pathways
    • Wnt
    • Nuclear
    • Epigenetics and Nuclear Signaling
    • Transcription
    • Transcription Factors
    • Cancer
    • Oncoproteins/suppressors
    • Tumor suppressors
    • PTC & Wnt pathway
    • Developmental Biology
    • Lineage specification
    • Mesoderm

Associated products

  • Alternative Versions

    • PE Anti-LEF1 antibody [EP2030Y] (ab210627)
    • Anti-LEF1 antibody [EP2030Y] - BSA and Azide free (ab227562)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
    • Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • Recombinant Protein

    • Recombinant Human LEF1 protein (ab132092)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab53293 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
ICC/IF
1/50 - 1/250.
Flow Cyt (Intra)
1/20.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

WB
1/5000. Detects a band of approximately 55 kDa (predicted molecular weight: 44 kDa).

For unpurified format use at 1/1000 dilution.

IHC-P (1)
1/50. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

Notes
ICC/IF
1/50 - 1/250.
Flow Cyt (Intra)
1/20.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

WB
1/5000. Detects a band of approximately 55 kDa (predicted molecular weight: 44 kDa).

For unpurified format use at 1/1000 dilution.

IHC-P
1/50. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

Target

  • Function

    Participates in the Wnt signaling pathway. Activates transcription of target genes in the presence of CTNNB1 and EP300. May play a role in hair cell differentiation and follicle morphogenesis. TLE1, TLE2, TLE3 and TLE4 repress transactivation mediated by LEF1 and CTNNB1. Regulates T-cell receptor alpha enhancer function. Binds DNA in a sequence-specific manner. PIAG antagonizes both Wnt-dependent and Wnt-independent activation by LEF1 (By similarity). Isoform 3 lacks the CTNNB1 interaction domain and may be an antagonist for Wnt signaling. Isoform 5 transcriptionally activates the fibronectin promoter, binds to and represses transcription from the E-cadherin promoter in a CTNNB1-independent manner, and is involved in reducing cellular aggregation and increasing cell migration of pancreatic cancer cells. Isoform 1 transcriptionally activates MYC and CCND1 expression and enhances proliferation of pancreatic tumor cells.
  • Tissue specificity

    Detected in thymus. Not detected in normal colon, but highly expressed in colon cancer biopsies and colon cancer cell lines. Expressed in several pancreatic tumors and weakly expressed in normal pancreatic tissue. Isoforms 1 and 5 are detected in several pancreatic cell lines.
  • Sequence similarities

    Belongs to the TCF/LEF family.
    Contains 1 HMG box DNA-binding domain.
  • Domain

    Proline-rich and acidic regions are implicated in the activation functions of RNA polymerase II transcription factors.
  • Cellular localization

    Nucleus. Found in nuclear bodies upon PIASG binding.
  • Target information above from: UniProt accession Q9UJU2 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 51176 Human
    • Omim: 153245 Human
    • SwissProt: Q9UJU2 Human
    • Unigene: 726506 Human
    • Alternative names

      • DKFZp586H0919 antibody
      • FLJ46390 antibody
      • LEF 1 antibody
      • LEF-1 antibody
      • Lef1 antibody
      • LEF1_HUMAN antibody
      • Lymphoid enhancer binding factor 1 antibody
      • Lymphoid enhancer-binding factor 1 antibody
      • T cell specific transcription factor 1 alpha antibody
      • T cell-specific transcription factor 1-alpha antibody
      • TCF 1 alpha antibody
      • TCF1 alpha antibody
      • TCF1-alpha antibody
      • TCF10 antibody
      • TCF1alpha antibody
      • TCF7L3 antibody
      • Transcription factor T cell specific 1 alpha antibody
      see all

    Images

    • Western blot - Anti-LEF1 antibody [EP2030Y] (ab53293)
      Western blot - Anti-LEF1 antibody [EP2030Y] (ab53293)
      All lanes : Anti-LEF1 antibody [EP2030Y] (ab53293) at 1/5000 dilution

      Lane 1 : Wild-type Jurkat cell lysate at 40 µg
      Lane 2 : Lef1 knockout Jurkat cell lysate at 40 µg
      Lane 3 : Jurkat cell lysate at 20 µg

      Performed under reducing conditions.

      Predicted band size: 44 kDa
      Observed band size: 40 kDa why is the actual band size different from the predicted?



      False colour image of Western blot: Anti-LEF1 antibody [EP2030Y] staining at 1/5000 dilution, shown in black; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab53293 was shown to bind specifically to LEF1. A band was observed at 40/53 kDa in wild-type Jurkat cell lysates with no signal observed at this size in Lef1 knockout cell line ab274898 (knockout cell lysate ab274956). To generate this image, wild-type and Lef1 knockout Jurkat cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times before development with Optiblot (ECL reagent ab133456) and imaged with 8 minutes exposure time. Secondary antibodies used were HRP conjugated Goat anti-Rabbit (H+L) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

    • Western blot - Anti-LEF1 antibody [EP2030Y] (ab53293)
      Western blot - Anti-LEF1 antibody [EP2030Y] (ab53293)
      Anti-LEF1 antibody [EP2030Y] (ab53293) at 1/5000 dilution + Jurkat (Human T cell leukemia T lymphocyte) whole cell lysates at 15 µg

      Secondary
      Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

      Predicted band size: 44 kDa
      Observed band size: 23-44 kDa why is the actual band size different from the predicted?



      This antibody recognizes all 7 LEF1 isoforms with the MW ranging from 23 to 44 KD based on immunogen blast.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LEF1 antibody [EP2030Y] (ab53293)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LEF1 antibody [EP2030Y] (ab53293)

      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human tonsil tissue sections labeling LEF1 with purified ab53293 at 1/50 dilution (1.94 µg/ml). Heat mediated antigen retrieval was performed using heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

    • Immunocytochemistry/ Immunofluorescence - Anti-LEF1 antibody [EP2030Y] (ab53293)
      Immunocytochemistry/ Immunofluorescence - Anti-LEF1 antibody [EP2030Y] (ab53293)

      Immunocytochemistry/ Immunofluorescence analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling LEF1 with purified ab53293 at 1/50 dilution (2.0 µg/ml). Cells were fixed in 100% Methanol. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) at 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

    • Flow Cytometry (Intracellular) - Anti-LEF1 antibody [EP2030Y] (ab53293)
      Flow Cytometry (Intracellular) - Anti-LEF1 antibody [EP2030Y] (ab53293)

      Intracellular Flow Cytometry analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling LEF1 with purified ab53293 at 1/20 dilution (10µg/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluorr® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue). 

       

       

    • Immunocytochemistry/ Immunofluorescence - Anti-LEF1 antibody [EP2030Y] (ab53293)
      Immunocytochemistry/ Immunofluorescence - Anti-LEF1 antibody [EP2030Y] (ab53293)

      ab53293 (unpurified) staining LEF1 in RAMOS (human Burkitt's lymphoma) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/500. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody at a dilution of 1/1000. DAPI was used as a nuclear counterstain and the negative control was PBS only.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LEF1 antibody [EP2030Y] (ab53293)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LEF1 antibody [EP2030Y] (ab53293)

      Paraffin-embedded human colon adenocarcinoma tissue stained for LEF1 using ab53293 (unpurified) at 1/50 dilution in immunohistochemical analysis.

       

    • Flow Cytometry (Intracellular) - Anti-LEF1 antibody [EP2030Y] (ab53293)
      Flow Cytometry (Intracellular) - Anti-LEF1 antibody [EP2030Y] (ab53293)

      Intracellular flow cytometric analysis of permeabilized Jurkat cells using ab53293 (unpurified) (red) or a rabbit IgG (negative) (green). 

       

       

    • Anti-LEF1 antibody [EP2030Y] (ab53293)
      Anti-LEF1 antibody [EP2030Y] (ab53293)

    Protocols

    To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    References (13)

    Publishing research using ab53293? Please let us know so that we can cite the reference in this datasheet.

    ab53293 has been referenced in 13 publications.

    • Tang L  et al. Long Noncoding RNA DCST1-AS1 Promotes Cell Proliferation and Metastasis in Triple-negative Breast Cancer by Forming a Positive Regulatory Loop with miR-873-5p and MYC. J Cancer 11:311-323 (2020). PubMed: 31897227
    • Zhao X  et al. Long Noncoding RNA NHEG1 Drives ß-Catenin Transactivation and Neuroblastoma Progression through Interacting with DDX5. Mol Ther 28:946-962 (2020). PubMed: 31982037
    • Hitomi Y  et al. NFKB1 and MANBA Confer Disease Susceptibility to Primary Biliary Cholangitis via Independent Putative Primary Functional Variants. Cell Mol Gastroenterol Hepatol 7:515-532 (2019). PubMed: 30528300
    • Zhang XF  et al. LEF-1 gene silencing inhibits pulmonary vascular remodeling and occurrence of pulmonary arterial hypertension through the ß-catenin signaling pathway. Biomed Pharmacother 108:817-827 (2018). PubMed: 30372893
    • Qiu J  et al. MiR-557 works as a tumor suppressor in human lung cancers by negatively regulating LEF1 expression. Tumour Biol 39:1010428317709467 (2017). PubMed: 28639890
    View all Publications for this product

    Customer reviews and Q&As

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

    1-5 of 5 Abreviews or Q&A

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-LEF1 antibody [EP2030Y]

    Average
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Pig Tissue sections (unerupted tooth buds)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: 10mM citric Acid monobasic pH6
    Permeabilization
    No
    Specification
    unerupted tooth buds
    Blocking step
    BSA as blocking agent for 20 minute(s) · Concentration: 5% · Temperature: 37°C
    Fixative
    10% Formalin
    Read More

    Abcam user community

    Verified customer

    Submitted Apr 27 2017

    Flow Cytometry abreview for Anti-LEF1 antibody [EP2030Y]

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Flow Cytometry
    Sample
    Human Cell (REH B cell)
    Specification
    REH B cell
    Fixation
    Paraformaldehyde
    Permeabilization
    Yes - saponin
    Read More

    Abcam user community

    Verified customer

    Submitted Apr 01 2010

    Question

    Dear Sir/Madam,
    I am using the Anti-LEF1 antibody [EP2030Y] (ab53293) for cytometry analysis and would like to know if it recognizes all the known LEF1 isoforms.
    Thank you in advance,

    Read More

    Abcam community

    Verified customer

    Asked on Apr 23 2012

    Answer

    Thank you for your inquiry.
    The exact immunogen sequence is proprietary information and I unfortunately do not have access to it.
    But the lab confirmed that all isoforms should be recognized by ab53293 since all isoforms contain the immunogen sequence with a 100% homology.
    This has not been tested experimentally, but a cross-reaction is very likely due to the perfect homology.
    I hope this information is helpful and wish you good luck with your research.

    Read More

    Abcam Scientific Support

    Answered on Apr 23 2012

    Question

    Dear Madam / Sir,

    A researher using ab53293 lef-1 has a band 70kDa; but It is written a expected band at app. 55kDa on the datasheet. According to uniport, lef-1 doesn't have any isoform with mass 70 kDa. Do you have any interpretation why It is detected at different mass? (Whether It is detected a different isoform of the protein or the antibody cross-reacts with a different protein mistakely).

    Researcher has performed ab53293 lef-1 western blot application on Jurkat and 293T cell lines.

    Read More

    Abcam community

    Verified customer

    Asked on Mar 19 2012

    Answer

    Thank you for taking the time to contact us. I am sorry to hearthe customer has some concerns regarding the results from this antibody.

    I would like to reassure you that this antibody is tested and covered by our 6 month guarantee for WB and human samples. In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

    With regards to the band size, according to the SwissProt/Uniprot protein database site, there are several smaller isoforms of LEF1, but no larger ones at 70 kDa. Could you confirm the customer is reducing and denaturing the samples and running them on a reducing gel? This will ensure the protein is in the correct conformation to run at the correct molecular weight.

    I would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily.

    I would appreciate if you could also provide an image (including molecular weight markers)which would help us to assess the results.

    Thank you for your time and cooperation. We look forward to receiving the completed questionnaire and image.

    Order Details
    Antibody code:

    Problem
    Choose: Non-specific band Multiple bands No signal or weak signal High background

    Lot number

    Purchase order number
    or preferably Abcam order number:



    General Information
    Antibody storage conditions (temperature/reconstitution etc)


    Description of the problem (high background, wrong band size, more bands, no band etc.)


    Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)


    Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)


    Amount of protein loaded


    Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)


    Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)


    Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


    Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


    Detection method (ECL, ECLPlus etc.)


    Positive and negative controls used (please specify)



    Optimization attempts (problem solving)
    How many times have you tried the Western?



    Have you run a "No Primary" control?
    Yes No

    Do you obtain the same results every time?
    Yes No
    e.g. are the background bands always in the same place?


    What steps have you altered?


    Additional Notes:


    Image:
    We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to asess the results.

    Read More

    Abcam Scientific Support

    Answered on Mar 19 2012

    Question

    I use the primary antibody(cat:ab53293) to detect lung fibroblast lef1 content in western blot. But I can't find any band whatever the concentration I used. I ask whether you have positive control sample? If you have, please send it to me.

    Read More

    Abcam community

    Verified customer

    Asked on Feb 24 2012

    Answer

    Thank you for contacting Abcam.

    I am sorry that you are having problems with ab53293 in western blot. If you are looking for a positive control then I would recommend ab7899:

    https://www.abcam.com/Jurkat-Human-T-cell-lymphoblast-like-cell-line-Whole-Cell-Lysate-ab7899.html



    Also I would be very happy to try and help you resolve your western blot issues, if you could answer the questions below, I will see if thereare any protocol changes that I can suggest:

    1 - What sample type are you using (including species)?

    2 - Are you seeing any bands, no bands etc?

    3 - How much sample are you loading?

    4 - What type of blocking agent are you using (eg milk, BSA etc)?

    5 - What primary antibody concentrations have you tried and how long do you incubate for?

    I look forward to your reply and helping you resolve this issue.

    Read More

    Abcam Scientific Support

    Answered on Feb 24 2012

    Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
    For licensing inquiries, please contact partnerships@abcam.com

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