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Immunology Adaptive Immunity T Cells CD
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RecombinantRabMAb

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

  • Datasheet
  • SDS
  • Certificate of Compliance
Reviews (1) Submit a question References (1)

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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)
  • 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

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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 & Species

    Application Species
    Flow Cyt
    Human
    ICC/IF
    Human
    IHC-P
    Human
    IP
    Human
    WB
    Human
    See all applications and species data
  • 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). 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

  • 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: 7.2
    Preservative: 0.01% Sodium azide
    Constituents: PBS, 40% Glycerol, 0.05% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EPR20261
  • Isotype

    IgG
  • Research areas

    • Immunology
    • Adaptive Immunity
    • T Cells
    • CD
    • Immunology
    • Cell Type Markers
    • CD
    • NK Cells
    • Immunology
    • Secreted Molecules
    • Other secreted molecules
    • Immunology
    • Adaptive Immunity
    • Regulatory T Cells

Associated products

  • Alternative Versions

    • PE Anti-LAG-3 antibody [EPR20261] (ab225484)
    • Alexa Fluor® 488 Anti-LAG-3 antibody [EPR20261] (ab225485)
    • Alexa Fluor® 647 Anti-LAG-3 antibody [EPR20261] (ab225486)
    • Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) (ab150079)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab97051)
  • Conjugation kits

    • PE / R-Phycoerythrin Conjugation Kit - Lightning-Link® (ab102918)
    • APC Conjugation Kit - Lightning-Link® (ab201807)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • Related Products

    • VeriBlot for IP Detection Reagent (HRP) (ab131366)
    • Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) (ab93684)

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.

Guaranteed

Tested applications are guaranteed to work and covered by our Abpromise guarantee.

Predicted

Predicted to work for this combination of applications and species but not guaranteed.

Incompatible

Does not work for this combination of applications and species.

Application Species
Flow Cyt
Human
ICC/IF
Human
IHC-P
Human
IP
Human
WB
Human
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.
  • Target information above from: UniProt accession P18627 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 3902 Human
    • Omim: 153337 Human
    • SwissProt: P18627 Human
    • Unigene: 409523 Human
    • 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 1/500 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.

       

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

    Protocols

    • Flow cytometry protocols
    • Immunoprecipitation protocols
    • Immunohistochemistry protocols
    • Immunocytochemistry & immunofluorescence protocols
    • Western blot protocols

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    Certificate of Compliance

    To download a Certificate of Compliance, please enter your Lot number below:

    References (1)

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

    ab209236 has been referenced in 1 publication.

    • 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

    Customer reviews and Q&As

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

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

    Good
    Abreviews
    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

    Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
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