Key Product Details

Validated by

Biological Validation

Species Reactivity

Validated:

Human

Cited:

Human

Applications

Validated:

Immunohistochemistry, Western Blot, Flow Cytometry, CyTOF-reported

Cited:

Western Blot, Flow Cytometry, Immunocytochemistry, Functional Assay

Label

Unconjugated

Antibody Source

Polyclonal Goat IgG
View all LAG-3 Primary Antibodies »

Product Specifications

Immunogen

Mouse myeloma cell line NS0-derived recombinant human LAG-3
Leu23-Leu450
Accession # P18627

Specificity

Detects human LAG-3 in direct ELISAs and Western blots. In direct ELISAs, less than 10% cross-reactivity with recombinant mouse LAG-3 is observed.

Clonality

Polyclonal

Host

Goat

Isotype

IgG

Scientific Data Images for Human LAG‑3 Antibody

Detection of Human LAG-3 antibody by Western Blot.

Detection of Human LAG‑3 by Western Blot.

Western blot shows lysates of human peripheral blood mononuclear cells (PBMC) untreated or treated (+) with 1 ug/mL PHA for 5 days and HDLM-2 human Hodgkin's lymphoma cell line. PVDF membrane was probed with 1 µg/mL of Goat Anti-Human LAG-3 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2319) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). Specific bands were detected for LAG-3 at approximately 60-75 kDa (as indicated). GAPDH (Catalog # MAB5718) is shown as a loading control. This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of LAG-3 antibody in CD3+Human PBMCs antibody by Flow Cytometry.

Detection of LAG‑3 in CD3+Human PBMCs by Flow Cytometry.

Human peripheral blood mononuclear cells (PBMCs) either (A) untreated or (B) treated with 1 ug/mL PHA for 5 days were stained with Goat Anti-Human LAG-3 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2319) followed by Allophycocyanin-conjugated Anti-Goat IgG Secondary Antibody (Catalog # F0108) and Mouse Anti-Human CD3e PE-conjugated Monoclonal Antibody (Catalog # FAB100P). Quadrant markers were set based on control antibody staining (Catalog # AB-108-C). View our protocol for Staining Membrane-associated Proteins.

Detection of LAG-3 antibody in HEK293 Human Cell Line Transfected with Human LAG-3 and eGFP antibody by Flow Cytometry.

Detection of LAG-3 in HEK293 Human Cell Line Transfected with Human LAG-3 and eGFP by Flow Cytometry.

HEK293 human embryonic kidney cell line transfected with either (A) human LAG-3 or (B) irrelevant transfectants and eGFP was stained with Goat Anti-Human LAG-3 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2319) followed by Allophycocyanin-conjugated Anti-Goat IgG Secondary Antibody (Catalog # F0108). Quadrant markers were set based on control antibody staining(Catalog # AB-108-C, data not shown). View our protocol for Staining Membrane-associated Proteins.

LAG‑3 in Human Spleen.

LAG‑3 was detected in immersion fixed paraffin-embedded sections of human spleen using Goat Anti-Human LAG‑3 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2319) at 3 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Goat IgG VisUCyte™ HRP Polymer Antibody (VC004). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (CTS013). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to lymphocytes. Staining was performed using our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

LAG‑3 in Mouse Spleen.

LAG‑3 was detected in immersion fixed paraffin-embedded sections of mouse spleen using Goat Anti-Human LAG‑3 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2319) at 15 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Goat IgG VisUCyte™ HRP Polymer Antibody (VC004). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (CTS013). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to lymphocytes. Staining was performed using our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

Applications for Human LAG‑3 Antibody

Application
Recommended Usage

CyTOF-reported

Lowther, D.E. et al. (2016) JCI Insight 1:e85935. Ready to be labeled using established conjugation methods. No BSA or other carrier proteins that could interfere with conjugation.

Flow Cytometry

0.25 µg/106 cells
Sample: CD3+ human peripheral blood mononuclear cells (PBMCs) treated with PHA and HEK293 Human Cell Line Transfected with Human LAG-3 and eGFP

Immunohistochemistry

3-15 µg/mL
Sample: Immersion fixed paraffin-embedded sections of human spleen and mouse spleen

Western Blot

1 µg/mL
Sample: Human peripheral blood mononuclear cells (PBMC) treated with PHA and HDLM‑2 human Hodgkin's lymphoma cell line

Flow Cytometry Panel Builder

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Flow Cytometry

Formulation, Preparation, and Storage

Purification

Antigen Affinity-purified

Reconstitution

Reconstitute at 0.2 mg/mL in sterile PBS. For liquid material, refer to CoA for concentration.

Formulation

Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.

Shipping

Lyophilized product is shipped at ambient temperature. Liquid small pack size (-SP) is shipped with polar packs. Upon receipt, store immediately at the temperature recommended below.

Stability & Storage

Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.

Calculators

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: LAG-3

LAG-3 (Lymphocyte activation gene-3), also known as CD223, is a member of the immunoglobulin superfamily (IgSF). The mature LAG-3 protein is a 496 amino acid (aa) membrane protein with a 421 aa extracellular region which contains four IgSF domains, a 21 aa transmembrane region and a 54 aa cytoplasmic region. LAG-3 and CD4 molecules share < 20% aa sequence homology but have a similar structure (1, 2). Both molecules bind to MHC class II. LAG-3 binds to MHC class II with higher affinity compared to CD4. Both LAG-3 and CD4 genes are located on the distal part of the short arm of chromosome 12. LAG-3 is an activation-induced molecule, expressed on activated T cells and NK cells, but not on resting T cells. Studies using LAG-3 -/- mice have shown significant delay of T cell apoptosis following antigen stimulation and increased size of memory T cells pool following infection (3, 4). It also has been reported that anti-LAG-3 antibodies up-regulate T cell activation by blocking interaction of LAG-3 and MHC class II. The study has demonstrated that LAG-3 is selectively expressed on activated CD4+CD25+ TReg cells and plays a role in their suppressive activity (5). This evidence indicated, unlike the interaction of CD4 with MHC class II that plays a positive role in T cell activation, LAG-3 binds to MHC class II and negatively regulates T cell activation through LAG-3 signaling. On the other hand, studies have shown that binding of LAG-3 to MHC class II molecules on antigen presenting cells induce maturation of dendritic cells and cytokine secretion by monocytes through MHC class II signal transduction (6). Taken together, LAG-3 may have two major functions, it negatively regulates T cells activation through LAG-3 signaling and stimulates antigen presenting cells which express MHC class II.

References

  1. Triebel, F. et al. (1990) J. Exp. Med. 171:1393.
  2. Baixeras, E. et al. (1992) J. Exp. Med 176:327.
  3. Workman, C.J. and D.A. Vignali (2003) Eur. J. Immunol. 33:970.
  4. Workman, C.J. et al. (2004) J. Immunol. 172:5450.
  5. Huang, C.T. et al. (2004) Immunity 21:503.
  6. Andreae, S. et al. (2003) Blood 102:2130.

Long Name

Lymphocyte-activation Gene 3

Alternate Names

CD223, LAG3

Entrez Gene IDs

3902 (Human); 16768 (Mouse); 297596 (Rat); 102122272 (Cynomolgus Monkey)

Gene Symbol

LAG3

UniProt

P18627

Additional LAG-3 Products

Product Documents for Human LAG‑3 Antibody

Certificate of Analysis

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Product Specific Notices for Human LAG‑3 Antibody

For research use only

Citations for Human LAG‑3 Antibody

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Protocols

Find general support by application which include: protocols, troubleshooting, illustrated assays, videos and webinars.

FAQs for Human LAG‑3 Antibody

Showing  1 - 2 of 2 FAQs Showing All

  • Q: Can this LAG-3 antibody be used in ELISA? 

    A: We have not validated this particular LAG-3 antibody for ELISA in mouse; however, it has been cited as being used as a detection antibody in a publicatoin (PMID 15946792).

  • Q: I'm using your LAG-3 antibody in western blot but the observed band is a different size than the predicted band size, do you know what the bands are?

    A: There are several alternative splice varients of LAG-3 that could explain the different sized proteins stained by the LAG-3 antibody. It is also believed that there are two secreted forms of LAG-3, one that is about 36 kDa and another at 52 kDa.

  • Q: Can this LAG-3 antibody be used in ELISA? 

    A: We have not validated this particular LAG-3 antibody for ELISA in mouse; however, it has been cited as being used as a detection antibody in a publicatoin (PMID 15946792).

  • Q: I'm using your LAG-3 antibody in western blot but the observed band is a different size than the predicted band size, do you know what the bands are?

    A: There are several alternative splice varients of LAG-3 that could explain the different sized proteins stained by the LAG-3 antibody. It is also believed that there are two secreted forms of LAG-3, one that is about 36 kDa and another at 52 kDa.

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