Recombinant
RabMAb

Recombinant Anti-LAG-3 antibody [EPR20261] (ab209236)

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Overview

  • Product name

    Anti-LAG-3 antibody [EPR20261]
    See all LAG-3 primary antibodies

  • Description

    Rabbit monoclonal [EPR20261] to LAG-3

  • Host species

    Rabbit

  • Tested applications

    Suitable for: WB, IHC-P, ICC/IF, Flow Cyt, IPmore details

  • Species reactivity

    Reacts with: Human

  • Immunogen

    Recombinant fragment within Human LAG-3 aa 1-300. The exact sequence is proprietary.
    Database link: P18627

  • Positive control

    • WB: Human LAG3 Fc chimera recombinant protein (aa23-450). IHC-P: Human tonsil and Hodgkin’s lymphoma tissues. ICC/IF: HEK-293T cells transfected with a GFP-tagged LAG3 expression construct. Flow Cyt: HEK-293T transfected with a GFP-tagged human LAG3 construct. IP: HEK-293T transfected with a GFP-tagged human LAG3 construct whole cell lysate.

  • General notes

    This product was previously labelled as Lymphocyte Activation Gene 3.

    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.

Properties

Applications

Our Abpromise guarantee covers the use of ab209236 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 90 kDa (predicted molecular weight: 57 kDa).
IHC-P 1/1000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Different mRNA expression levels of LAG3 in brain have been reported in the literature (PMID: 1692078; PMID: 12825348). In IHC, under our experimental conditions, this antibody showed no positive staining on human cerebral cortex.
ICC/IF 1/100.
Flow Cyt 1/500.
IP 1/30.

Target

  • Function

    Involved in lymphocyte activation. Binds to HLA class-II antigens.

  • Tissue specificity

    On cell surface of activated NK and T-lymphocytes.

  • Sequence similarities

    Contains 3 Ig-like C2-type (immunoglobulin-like) domains.
    Contains 1 Ig-like V-type (immunoglobulin-like) domain.

  • Cellular localization

    Membrane.
  • Information by UniProt

  • Database links

    • Alternative names

      • CD223 antibody
      • CD223 antigen antibody
      • FDC protein antibody
      • LAG-3 antibody
      • Lag3 antibody
      • LAG3_HUMAN antibody
      • Lymphocyte activating 3 antibody
      • Lymphocyte activation gene 3 antibody
      • Lymphocyte activation gene 3 protein antibody
      • Protein FDC antibody
      see all

    Images

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [EPR20261] (ab209236)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [EPR20261] (ab209236)

      Immunohistochemical analysis of paraffin-embedded human tonsil Hodgkin’s lymphoma labeling LAG-3 with ab209236 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on immunocytes of the human Hodgkin’s lymphoma [PMID: 11527700; PMID: 16757686].

      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.

    • Immunocytochemistry/ Immunofluorescence - Anti-LAG-3 antibody [EPR20261] (ab209236)
      Immunocytochemistry/ Immunofluorescence - Anti-LAG-3 antibody [EPR20261] (ab209236)

      Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293T (Human epithelial cell line from embryonic kidney) cells transfected with GFP-tagged LAG3 expression construct or GFP only, labeling LAG-3 with ab209236 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 647) (ab150079) secondary antibody at 1/1000 dilution (green).

      Confocal image showing positive staining on HEK-293T cells transfected with a GFP-tagged LAG3 expression construct.

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

      Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor® 647) (ab150079) at 1/1000 dilution.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [EPR20261] (ab209236)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [EPR20261] (ab209236)

      Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling LAG-3 with ab209236 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Sporadic cytoplasmic staining on immunocytes of human tonsil [PMID: 11527700; PMID: 16757686].

      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.

    • Flow Cytometry - Anti-LAG-3 antibody [EPR20261] (ab209236)
      Flow Cytometry - Anti-LAG-3 antibody [EPR20261] (ab209236)

      Flow cytometric analysis of Human peripheral blood mononuclear cells treated with 1 μg/mL PHA for 3 days cells with ab209236 at 1/50 dilution (right) compared with a rabbit monoclonal IgG isotype control (ab172730; left). ab150077 at 1/2000 dilution was used as the secondary antibody.

      Only the CD3+ population are also positive for LAG3. Gated on total viable cells.

       

    • Flow Cytometry - Anti-LAG-3 antibody [EPR20261] (ab209236)
      Flow Cytometry - Anti-LAG-3 antibody [EPR20261] (ab209236)

      Flow cytometric analysis of HEK-293T (Human epithelial cell line from embryonic kidney) cells transfected with a GFP-tagged human LAG-3 construct labeling LAG-3 with ab209236 at 1/500 dilution (right) compared with a rabbit monoclonal IgG isotype control (ab172730; left). Goat anti rabbit IgG (Alexa Fluor® 647) ab150079 at 1/2000 dilution was used as the secondary antibody.

      Note: Fresh cells without fixation and permeabilization were used to perform FC testing. Only GFP positive population results in LAG3 positive staining (Q2, right panel).

    • Western blot - Anti-LAG-3 antibody [EPR20261] (ab209236)
      Western blot - Anti-LAG-3 antibody [EPR20261] (ab209236)
      Anti-LAG-3 antibody [EPR20261] (ab209236) at 1/1000 dilution + Human LAG3 Fc chimera recombinant protein (aa23-450) at 0.01 µg

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

      Predicted band size: 57 kDa
      Observed band size: 90 kDa
      why is the actual band size different from the predicted?


      Exposure time: 1 second


      Blocking/Dilution buffer: 5% NFDM/TBST.

    • Immunoprecipitation - Anti-LAG-3 antibody [EPR20261] (ab209236)
      Immunoprecipitation - Anti-LAG-3 antibody [EPR20261] (ab209236)

      LAG-3 was immunoprecipitated from 0.35 mg of HEK-293T (Human epithelial cell line from embryonic kidney) transfected with a GFP-tagged human LAG3 construct whole cell lysate with ab209236 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab209236 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

      Lane 1: HEK-293T transfected with a GFP-tagged human LAG3 construct whole cell lysate 10 μg (Input).

      Lane 2: ab209236 IP in HEK-293T transfected with a GFP-tagged human LAG3 construct whole cell lysate.

      Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab209236 in HEK-293T transfected with a GFP-tagged human LAG3 construct whole cell lysate.

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

      Exposure time: 1 second.

       

    References

    ab209236 has not yet been referenced specifically in any publications.

    Publishing research using ab209236? Please let us know so that we can cite the reference in this datasheet.

    Customer reviews and Q&As

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    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-Lymphocyte Activation Gene 3 antibody [EPR20261]

     Good
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Human Tissue sections (tonsil)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: TRIS-EDTA Buffer pH 9,0
    Permeabilization
    Yes - Wash Buffer from Dako with Tween
    Specification
    tonsil
    Fixative
    Paraformaldehyde
    Read More

    Mr. Rudolf Jung

    Verified customer

    Submitted Jul 25 2018

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