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 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 CD3 epsilon 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 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) irrelelvant 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.
Preparation and Storage
Reconstitute at 0.2 mg/mL in sterile PBS.
The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below. *Small pack size (SP) is shipped with polar packs. Upon receipt, store it immediately at -20 to -70 °C
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.
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.
Triebel, F. et al. (1990) J. Exp. Med. 171:1393.
Baixeras, E. et al. (1992) J. Exp. Med 176:327.
Workman, C.J. and D.A. Vignali (2003) Eur. J. Immunol. 33:970.
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