Recombinant

Recombinant Anti-LAG-3 antibody [CAL25] (ab237718)

Overview

  • Product name

    Anti-LAG-3 antibody [CAL25]
    See all LAG-3 primary antibodies
  • Description

    Rabbit monoclonal [CAL25] 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 considered to be commercially sensitive.

  • Positive control

    • IHC-P: Human tonsil tissue. WB: HEK-293T transfected with GFP-tagged LAG-3 expression vector whole cell lysate. Human peripheral blood mononuclear cell (PBMC) treated with 10µg/ml PHA for 48h whole cell lysate. Flow: Human peripheral blood mononuclear cell (PBMC) treated with 10µg/ml PHA for 48h. IP:HEK-293T transfected with GFP-tagged LAG-3 expression vector whole cell lysate.
  • General notes

    This product was previously labelled as Lymphocyte Activation Gene 3.

Properties

Applications

Our Abpromise guarantee covers the use of ab237718 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. Predicted molecular weight: 57 kDa.
Flow Cyt 1/50.
IP 1/30.
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.
    • 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

    • Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling LAG-3 using ab237718 at 1/800 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining on the human tonsil is observed. The section was incubated with ab237718 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

      Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP). 

    • LAG-3 was immunoprecipitated from 0.35 mg HEK-293T (human embryonic kidney epithelial cell) transfected with GFP-tagged LAG-3 expression vector whole cell lysate using ab237718 at 1/30 dilution. Western blot was performed on the immunoprecipitate using ab237718 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used as the secondary antibody at 1/5000 dilution.

      Lane 1: HEK-293T  transfected with GFP-tagged LAG-3 expression vector whole cell lysate 10 µg (input).
      Lane 2: ab237718 IP in HEK-293T GFP-tagged LAG-3 expression vector whole cell lysate.
      Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab237718 in 293T transfected with GFP-tagged LAG-3 expression vector whole cell lysate.

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

      Exposure time: 30 seconds.

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

    • Flow cytometric analysis of 2% paraformaldehyde-fixed 0.1% Tween-20 permeabilized Human peripheral blood mononuclear cells (PBMC) treated with 10μg/ml PHA for 48h labeling LAG-3 with ab237718 at 1/50 dilution (Right) compared to a Rabbit monoclonal IgG (ab172730, Left). Goat anti rabbit IgG (Alexa Fluor® 488, ab150097) was used as the secondary at 1/5000 dilution.

      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 ab237718 (Right).

    • All lanes : Anti-LAG-3 antibody [CAL25] (ab237718) at 1/1000 dilution

      Lane 1 : HEK-293T (human embryonic kidney epithelial cell) whole cell lysate
      Lane 2 : HEK-293T transfected with GFP-tagged LAG-3 expression vector 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: 70, 16 kDa why is the actual band size different from the predicted?


      Exposure time: 3 minutes


      Blocking/diluting buffer and concentration: 5% NFDM/TBST

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

    • All lanes : Anti-LAG-3 antibody [CAL25] (ab237718) 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 48h 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: 70, 16 kDa why is the actual band size different from the predicted?


      Exposure time: 3 minutes


      Blocking buffer and concentration: 5% NFDM/TBST

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

       

    • Formalin-fixed, paraffin-embedded human tonsil tissue stained for LAG-3 using ab237718 at 0.3 μg/ml in immunohistochemical analysis.

    References

    ab237718 has not yet been referenced specifically in any publications.

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