LAG-3 Antibody [Unconjugated]

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." > 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." title="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." />

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 # <a class=

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." > 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." title="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." />

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 # <a class=

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." > 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." title="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." />

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 # <a class=

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." > 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." title="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 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 (<a class=

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." > 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." title="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." />

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 (<a class=

• Reactivity: Hu
• Applications: WB, Flow, IHC, CyTOF-ready
• Clonality: Polyclonal
• Host: Goat
• Conjugate: Unconjugated
• Concentration: LYOPH

LAG-3 Antibody [Unconjugated] Summary

• 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.
• Source: N/A
• Isotype: IgG
• Clonality: Polyclonal
• Host: Goat
• Gene: LAG3
• Purity Statement: Antigen Affinity-purified

Applications/Dilutions

• Dilutions:
  • CyTOF-reported
  • Flow Cytometry 0.25 ug/10^6 cells
  • Immunohistochemistry 3-15 ug/mL
  • Western Blot 1 ug/mL

Packaging, Storage & Formulations

• 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.
• Buffer: 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.
• Preservative: No Preservative
• Concentration: LYOPH
• Reconstitution Instructions: Reconstitute at 0.2 mg/mL in sterile PBS.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for LAG-3 Antibody [Unconjugated]

• CD223 antigen
• CD223
• LAG3
• LAG-3
• lymphocyte activating 3
• lymphocyte activation gene 3 protein
• lymphocyte-activation gene 3
• Secreted lymphocyte activation gene 3 protein
• sLAG-3

Background

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⁺ T_Reg 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.

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.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for LAG-3 Antibody (AF2319)(5)

Publications using AF2319

• W Scheper, S Kelderman, LF Fanchi, C Linnemann, G Bendle, MAJ de Rooij, C Hirt, R Mezzadra, M Slagter, K Dijkstra, RJC Kluin, P Snaebjorns, K Milne, BH Nelson, H Zijlmans, G Kenter, EE Voest, JBAG Haanen, TN Schumacher Low and variable tumor reactivity of the intratumoral TCR repertoire in human cancers Nat. Med., 2018-12-03;0(0):. 2018-12-03 [PMID: 30510250] (Flow Cytometry, Human)
• X Lu, JW Horner, E Paul, X Shang, P Troncoso, P Deng, S Jiang, Q Chang, DJ Spring, P Sharma, JA Zebala, DY Maeda, YA Wang, RA DePinho Effective combinatorial immunotherapy for castration-resistant prostate cancer Nature, 2017-03-20;543(7647):728-732. 2017-03-20 [PMID: 28321130] (Functional Assay, Human)
• Fine needle aspirate flow cytometric phenotyping characterizes immunosuppressive nature of the mesothelioma microenvironment Sci Rep, 2016-08-19;6(0):31745. 2016-08-19 [PMID: 27539742] (Flow Cytometry, Human)
• Bae J, Lee S, Park C, Lee Y, Chun T Trafficking of LAG-3 to the surface on activated T cells via its cytoplasmic domain and protein kinase C signaling. J Immunol, 2014-08-08;193(6):3101-12. 2014-08-08 [PMID: 25108024] (Functional Assay, ICC, Western Blot, Human)
• Porichis F, Kwon DS, Zupkosky J, Tighe DP, McMullen A, Brockman MA, Pavlik DF, Rodriguez-Garcia M, Pereyra F, Freeman GJ, Kavanagh DG, Kaufmann DE Responsiveness of HIV-specific CD4 T cells to PD-1 blockade. Blood, 2011-06-07;118(4):965-74. 2011-06-07 [PMID: 21652684] (Flow Cytometry, Human)

FAQs for LAG-3 Antibody (AF2319). (Showing 1 - 2 of 2 FAQs).

1. Can this LAG-3 antibody be used in ELISA? 
   • 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).
2. 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?
   • 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.

Bioinformatics

• Gene Symbol: LAG3
• Uniprot:
  • P18627()