RecombinantRabMAb

Recombinant Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)

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Western blot - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)
  • Flow Cytometry - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)
  • Immunocytochemistry/ Immunofluorescence - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)
  • Flow Cytometry - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)
  • Immunoprecipitation - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)
  • Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)

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 - BSA and Azide free
  • Suitable for: IHC-P, WB, ICC/IF, IP, Flow Cyt
  • Reacts with: Human

Overview

  • Product name

    Anti-LAG-3 antibody [EPR20261] - BSA and Azide free
    See all LAG-3 primary antibodies

  • Description

    Rabbit monoclonal [EPR20261] to LAG-3 - BSA and Azide free

  • Host species

    Rabbit

  • Tested applications

    Suitable for: IHC-P, WB, ICC/IF, IP, Flow Cytmore details

  • Species reactivity

    Reacts with: Human

  • Immunogen

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

  • Positive control

    • WB: HDLM-2 cells and Human LAG-3 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 LAG-3 construct. IP: HEK-293T transfected with a GFP-tagged human LAG-3 construct whole cell lysate.

  • General notes

    ab227579 is the carrier-free version of ab209236 This format is designed for use in antibody labeling, including fluorochromes, metal isotopes, oligonucleotides, enzymes.

     

    Our carrier-free formats are supplied in a buffer free of BSA, sodium azide and glycerol for higher conjugation efficiency.

    Use our conjugation kits  for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

    Ab227579 is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm.

    Maxpar® is a trademark of Fluidigm Canada Inc.

    This product was previously labelled as Lymphocyte Activation Gene 3.

    Reproducibility is key to advancing scientific discovery and accelerating scientists’ next breakthrough.

    Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility.

    We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee.

    In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.

    We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.

    Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.

    Please check that this product meets your needs before purchasing. If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, as well as customer reviews and Q&As.

Properties

Applications

Our Abpromise guarantee covers the use of ab227579 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
IHC-P Use at an assay dependent concentration. 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.
WB Use at an assay dependent concentration. Detects a band of approximately 90 kDa (predicted molecular weight: 57 kDa).
ICC/IF Use at an assay dependent concentration.
IP Use at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.

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] - BSA and Azide free (ab227579)
      Western blot - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)
      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: 57 kDa



      This IHC data was generated using the same anti-LAG-3 antibody clone, EPR20261, in a different buffer formulation (cat# ab209236).

      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

    • Flow Cytometry - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)
      Flow Cytometry - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)

      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 LAG-3. Gated on total viable cells.

       

      This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab209236).

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)

      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.

      This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab209236).

      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] - BSA and Azide free (ab227579)
      Immunocytochemistry/ Immunofluorescence - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)

      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 LAG-3 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.

      This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab209236).

    • Flow Cytometry - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)
      Flow Cytometry - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)

      Flow cytometric analysis of HEK-293T (Human epithelial cell line from embryonic kidney) cells transfected with a GFP-tagged human LAG3 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).

      This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab209236).

    • Immunoprecipitation - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)
      Immunoprecipitation - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)

      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.

       

      This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab209236).

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)

      This IHC data was generated using the same anti-LAG-3 antibody clone, EPR20261, in a different buffer formulation (cat# 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.

    • Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)
      Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)

    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

    References (0)

    Publishing research using ab227579? Please let us know so that we can cite the reference in this datasheet.

    ab227579 has not yet been referenced specifically in any publications.

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