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RecombinantRabMAb

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

Reviews (1) Submit a question References (10)
Western blot - Anti-LAG-3 antibody [EPR20261] (ab209236)
  • Western blot - Anti-LAG-3 antibody [EPR20261] (ab209236)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [EPR20261] (ab209236)
  • Immunocytochemistry/ Immunofluorescence - Anti-LAG-3 antibody [EPR20261] (ab209236)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [EPR20261] (ab209236)
  • Flow Cytometry - Anti-LAG-3 antibody [EPR20261] (ab209236)
  • Flow Cytometry - Anti-LAG-3 antibody [EPR20261] (ab209236)
  • Western blot - Anti-LAG-3 antibody [EPR20261] (ab209236)
  • Immunoprecipitation - Anti-LAG-3 antibody [EPR20261] (ab209236)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [EPR20261] (ab209236)
  • Anti-LAG-3 antibody [EPR20261] (ab209236)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR20261] to LAG-3
  • Suitable for: WB, IHC-P, ICC/IF, Flow Cyt, IP
  • Reacts with: Human

Conjugates logo Related conjugates and formulations

Alexa Fluor® 488 Alexa Fluor® 555 Alexa Fluor® 594 Alexa Fluor® 647 Carrier Free PE

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

  • Positive control

    • WB: HDLM-2 cells and Human LAG3 Fc chimera recombinant protein (aa23-450). 293T transfected with LAG3 expression vector containing a GFP tag, whole cell lysate. 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 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

The Abpromise guarantee

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
1/500. Detects a band of approximately 70 kDa (predicted molecular weight: 54 kDa).
IHC-P (1)
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.
Notes
WB
1/500. Detects a band of approximately 70 kDa (predicted molecular weight: 54 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

    • Western blot - Anti-LAG-3 antibody [EPR20261] (ab209236)
      Western blot - Anti-LAG-3 antibody [EPR20261] (ab209236)
      All lanes : Anti-LAG-3 antibody [EPR20261] (ab209236) at 1/5000 dilution

      Lane 1 : 293T (Human embryonic kidney epithelial cell) transfected with an empty vector (vector control), containing a GFP tag, whole cell lysate
      Lane 2 : 293T transfected with LAG3 expression vector containing a GFP tag, whole cell lysate

      Lysates/proteins at 15 µg per lane.

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

      Predicted band size: 54 kDa


      Exposure time: 5 seconds


      Blocking/Diluting buffer: 5% NFDM/TBST.

    • Western blot - Anti-LAG-3 antibody [EPR20261] (ab209236)
      Western blot - Anti-LAG-3 antibody [EPR20261] (ab209236)
      All lanes : Anti-LAG-3 antibody [EPR20261] (ab209236) at 1/500 dilution

      Lane 1 : HDLM-2 (Human Hodgkin lymphoma ) whole cell lysate
      Lane 2 : Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution (Goat anti-Rabbit IgG H&L (IRDye® 800RCW) preadsorbed)

      Predicted band size: 54 kDa



      Primary loading control and concentration: Anti-GAPDH antibody [6C5] - Loading Control (ab8245) at 1/20000 dilution 

      Secondary loading control and concentration: Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) at 1/10000 dilution

      Lanes 1-2: Merged signal (red and green). Green – ab209236 observed at 54-70 kDa. Red - loading control ab8245 observed at 36 kDa.
      ab209236 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800RCW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

      The expression profile observed in Jurkat is consistent with the literature (PMID: 25108024).

      Negative control: Jurkat (PMID: 25108024)

       Observed MW: 54-70 kDa

    • 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: 54 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.

       

    • 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)

      Tissue Microarrays stained for "Anti-LAG-3 antibody [EPR20261]” using "ab209236" in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negative (cross mark) staining per sample type tested. The sections were pre-treated using Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). The sections were incubated with ab209236 at +4°C overnight followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer).

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

    References (10)

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

    ab209236 has been referenced in 10 publications.

    • Chen WJ  et al. Heterogeneity of tumor microenvironment is associated with clinical prognosis of non-clear cell renal cell carcinoma: a single-cell genomics study. Cell Death Dis 13:50 (2022). PubMed: 35017463
    • Liu X  et al. Single-cell transcriptomics links malignant T cells to the tumor immune landscape in cutaneous T cell lymphoma. Nat Commun 13:1158 (2022). PubMed: 35241665
    • Zhang Q  et al. USP35 is a Potential Immunosuppressive Factor in Skin Cutaneous Melanoma. J Inflamm Res 15:3065-3082 (2022). PubMed: 35637872
    • Liu L  et al. Single cell sequencing reveals that CD39 inhibition mediates changes to the tumor microenvironment. Nat Commun 13:6740 (2022). PubMed: 36347860
    • Luo JW  et al. Differences in Immunological Landscape between EGFR-Mutated and Wild-Type Lung Adenocarcinoma. Dis Markers 2021:3776854 (2021). PubMed: 34484468
    • Gusak A  et al. Immunosuppressive Microenvironment and Efficacy of PD-1 Inhibitors in Relapsed/Refractory Classic Hodgkin Lymphoma: Checkpoint Molecules Landscape and Macrophage Populations. Cancers (Basel) 13:N/A (2021). PubMed: 34830831
    • Xie X  et al. The Expression Pattern and Clinical Significance of the Immune Checkpoint Regulator VISTA in Human Breast Cancer. Front Immunol 11:563044 (2020). PubMed: 33250890
    • Chen L  et al. Targeting CMTM6 Suppresses Stem Cell-Like Properties and Enhances Antitumor Immunity in Head and Neck Squamous Cell Carcinoma. Cancer Immunol Res 8:179-191 (2020). PubMed: 31771985
    • Hu J  et al. Single-Cell Transcriptome Analysis Reveals Intratumoral Heterogeneity in ccRCC, which Results in Different Clinical Outcomes. Mol Ther 28:1658-1672 (2020). PubMed: 32396851
    • Wang H  et al. Altered expression of TIM-3, LAG-3, IDO, PD-L1, and CTLA-4 during nimotuzumab therapy correlates with responses and prognosis of oral squamous cell carcinoma patients. J Oral Pathol Med 48:669-676 (2019). PubMed: 31132187

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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
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    MR. Rudolf Jung

    Verified customer

    Submitted Jul 25 2018

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