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    lag-3-antibody-cal26-ab237719.pdf

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Immunology Adaptive Immunity T Cells CD
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RecombinantRabMAb

Recombinant Anti-LAG-3 antibody [CAL26] (ab237719)

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Western blot - Anti-LAG-3 antibody [CAL26] (ab237719)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [CAL26] (ab237719)
  • Immunoprecipitation - Anti-LAG-3 antibody [CAL26] (ab237719)
  • Flow Cytometry - Anti-LAG-3 antibody [CAL26] (ab237719)
  • Western blot - Anti-LAG-3 antibody [CAL26] (ab237719)
  • Western blot - Anti-LAG-3 antibody [CAL26] (ab237719)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [CAL26] (ab237719)
  • Anti-LAG-3 antibody [CAL26] (ab237719)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [CAL26] to LAG-3
  • Suitable for: WB, Flow Cyt, IP, IHC-P
  • Reacts with: Human

Conjugates logo Related conjugates and formulations

Carrier Free

Overview

  • Product name

    Anti-LAG-3 antibody [CAL26]
    See all LAG-3 primary antibodies
  • Description

    Rabbit monoclonal [CAL26] to LAG-3
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, Flow Cyt, IP, IHC-Pmore details
    Unsuitable for: ICC/IF
  • Species reactivity

    Reacts with: Human
  • Immunogen

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

  • Positive control

    • WB: LAG-3-GFP transfected HEK-293 whole cell lysate; human PBMC whole cell lysate. IHC-P: Human tonsil tissue. Flow cyt: Human PBMCs treated with 10µg/ml PHA for 48 hours. IP: LAG-3-GFP transfected HEK-203& whole cell lysate.

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: 7.50
    Preservative: 0.05% Sodium azide
    Constituent: PBS
  • Concentration information loading...
  • Purity

    Protein A purified
  • Purification notes

    Purity >99%.
  • Clonality

    Monoclonal
  • Clone number

    CAL26
  • 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

    • Anti-LAG-3 antibody [CAL26] - BSA and Azide free (ab251605)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG Fc (Alexa Fluor® 488) preadsorbed (ab150097)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab97051)
  • Immunohistochemistry reagents

    • Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) (ab93684)
  • Isotype control

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

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab237719 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 16, 70 kDa (predicted molecular weight: 57 kDa).
Flow Cyt
1/250.
IP
1/20.
IHC-P
Use a concentration of 0.3 µg/ml. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Notes
WB
1/1000. Detects a band of approximately 16, 70 kDa (predicted molecular weight: 57 kDa).
Flow Cyt
1/250.
IP
1/20.
IHC-P
Use a concentration of 0.3 µg/ml. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Application notes
Is unsuitable for ICC/IF.

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 [CAL26] (ab237719)
      Western blot - Anti-LAG-3 antibody [CAL26] (ab237719)
      All lanes : Anti-LAG-3 antibody [CAL26] (ab237719) at 1/1000 dilution

      Lane 1 : HDLM-2 cell lysate
      Lane 2 : Jurkat cell lysate

      Lysates/proteins at 20 µg per lane.

      Performed under reducing conditions.

      Predicted band size: 57 kDa
      Observed band size: 57,70 kDa why is the actual band size different from the predicted?



      False colour image of Western blot: Anti-LAG-3 antibody [CAL26] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab237719 was shown to bind specifically to LAG-3. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution 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 then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [CAL26] (ab237719)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [CAL26] (ab237719)

      Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling LAG-3 with ab237719 at 1/400 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining on the human tonsil is observed. 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.

      Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

    • Immunoprecipitation - Anti-LAG-3 antibody [CAL26] (ab237719)
      Immunoprecipitation - Anti-LAG-3 antibody [CAL26] (ab237719)

      LAG-3 was immunoprecipitated from 0.35 mg of LAG-3-GFP transfected HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen)  whole cell lysate with ab237719 at 1/20 dilution. Western blot was performed from the immunoprecipitate using ab237719 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used as secondary antibody at 1/5000 dilution.

      Lane 1: LAG-3-GFP transfected HEK-293T whole cell lysate 10 μg (Input).
      Lane 2: ab237719 IP in LAG-3-GFP transfected HEK-293T whole cell lysate.
      Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab237719 in LAG-3-GFP transfected HEK-293T whole cell lysate.

      Blocking and dilution buffer and concentration: 5% NFDM/TBST.
      Exposure time: 30 seconds.

    • Flow Cytometry - Anti-LAG-3 antibody [CAL26] (ab237719)
      Flow Cytometry - Anti-LAG-3 antibody [CAL26] (ab237719)

      Flow cytometric analysis of 2% paraformaldehyde-fixed, 0.1% Tween-20 permeabilized human peripheral blood mononuclear cells ((PBMC) (treated with 10μg/ml PHA for 48 hours) cell line labeling LAG-3 with ab237719 at 1/250 dilution (Right) compared with Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (Left).

      Goat Anti-Rabbit IgG Fc (Alexa Fluor® 488) preadsorbed (ab150097), at 1/5000 dilution was used as the secondary antibody.

      Cells were surface stained with anti-CD3 conjugated to Alexa Fluor® 647. Then fixed with 2% PFA for 10min followed by intracellular staining rabbit IgG (Left) or ab237719 (Right).

    • Western blot - Anti-LAG-3 antibody [CAL26] (ab237719)
      Western blot - Anti-LAG-3 antibody [CAL26] (ab237719)
      All lanes : Anti-LAG-3 antibody [CAL26] (ab237719) at 1/1000 dilution

      Lane 1 : Untransfected HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
      Lane 2 : LAG-3-GFP overexpression HEK-293T whole cell lysate

      Lysates/proteins at 20 µg per lane.

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

      Predicted band size: 57 kDa
      Observed band size: 16,70 kDa why is the actual band size different from the predicted?


      Exposure time: 3 minutes


      Blocking and dilution buffer: 5% NFDM/TBST.

      This antibody also detects a 16kDa cleaved fragment. (PMID: 15557174).

      This blot was developed using a higher sensitivity ECL substrate.

    • Western blot - Anti-LAG-3 antibody [CAL26] (ab237719)
      Western blot - Anti-LAG-3 antibody [CAL26] (ab237719)
      All lanes : Anti-LAG-3 antibody [CAL26] (ab237719) at 1/1000 dilution

      Lane 1 : Human peripheral blood mononuclear cell (PBMC), whole cell lysate
      Lane 2 : Human peripheral blood mononuclear cell (PBMC) treated with 10µg/ml PHA for 48 hours, whole cell lysate
      Lane 3 : Human lymphoma lysate

      Lysates/proteins at 20 µg per lane.

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

      Predicted band size: 57 kDa
      Observed band size: 16,70 kDa why is the actual band size different from the predicted?


      Exposure time: 3 minutes


      Blocking and dilution buffer: 5% NFDM/TBST.

      This antibody also detects a 16kDa cleaved fragment. (PMID: 15557174).

      This blot was developed using a higher sensitivity ECL substrate.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [CAL26] (ab237719)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [CAL26] (ab237719)

      Formalin-fixed, paraffin-embedded human tonsil tissue stained for LAG-3 using ab237719 at 0.3 μg/ml in immunohistochemical analysis.

    • Anti-LAG-3 antibody [CAL26] (ab237719)
      Anti-LAG-3 antibody [CAL26] (ab237719)

    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

    • Datasheet download

      Download

    References (0)

    Publishing research using ab237719? Please let us know so that we can cite the reference in this datasheet.

    ab237719 has not yet been referenced specifically in any publications.

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