Anti-LAG-3 antibody [EPR20261] - Carrier free (ab227579)

Anti-LAG-3 [EPR20261] antibody (ab227579) is a rabbit monoclonal antibody that is provided in a PBS only formulation free from BSA and azide. Formulated to be conjugation-ready. It is used to detect LAG-3 in Western Blot, Flow Cytometry, IP, IHC-P, ICC/IF. Suitable for Human samples.

- BSA, sodium azide, and glycerol-free for consistent conjugation with fluorochromes, enzymes and more

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Related conjugates and formulations

ab209236
Unconjugated
Anti-LAG-3 antibody [EPR20261]
ab225485
Conjugated
Alexa Fluor® 488 Anti-LAG-3 antibody [EPR20261]
ab225486
Conjugated
Alexa Fluor® 647 Anti-LAG-3 antibody [EPR20261]
ab283627
Conjugated
Alexa Fluor® 594 Anti-LAG-3 antibody [EPR20261]
ab280858
Conjugated
Alexa Fluor® 555 Anti-LAG-3 antibody [EPR20261]
ab225484
Conjugated
PE Anti-LAG-3 antibody [EPR20261]

Images

Flow Cytometry - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (AB227579), expandable thumbnail

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Constituents: PBS
Form: Liquid
Clonality: Monoclonal

Immunogen

The exact immunogen used to generate this antibody is proprietary information.

Reactivity data

Select an application

Product promiseTestedExpectedPredictedNot recommended

	IP	Flow Cyt	WB	ICC/IF	IHC-P
Human	Tested	Tested	Tested	Tested	Tested

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info -	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info -	Notes Different mRNA expression levels of LAG3 in brain have been reported in the literature (PMID: 1692078; PMID: 12825348). In IHC, under our experimental conditions, this antibody showed no positive staining on human cerebral cortex. Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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Target data

See full target information LAG3

Function

Lymphocyte activation gene 3 protein: Inhibitory receptor on antigen activated T-cells (PubMed:20421648, PubMed:7805750, PubMed:8647185). Delivers inhibitory signals upon binding to ligands, such as FGL1 (By similarity). FGL1 constitutes a major ligand of LAG3 and is responsible for LAG3 T-cell inhibitory function (By similarity). Following TCR engagement, LAG3 associates with CD3-TCR in the immunological synapse and directly inhibits T-cell activation (By similarity). May inhibit antigen-specific T-cell activation in synergy with PDCD1/PD-1, possibly by acting as a coreceptor for PDCD1/PD-1 (By similarity). Negatively regulates the proliferation, activation, effector function and homeostasis of both CD8(+) and CD4(+) T-cells (PubMed:20421648, PubMed:7805750, PubMed:8647185). Also mediates immune tolerance: constitutively expressed on a subset of regulatory T-cells (Tregs) and contributes to their suppressive function (By similarity). Also acts as a negative regulator of plasmacytoid dendritic cell (pDCs) activation (By similarity). Binds MHC class II (MHC-II); the precise role of MHC-II-binding is however unclear (PubMed:8647185). Secreted lymphocyte activation gene 3 protein. May function as a ligand for MHC class II (MHC-II) on antigen-presenting cells (APC), promoting APC activation/maturation and driving Th1 immune response.

Alternative names

CD223, FDC, LAG3, Lymphocyte activation gene 3 protein, LAG-3

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Constituents: PBS
Form: Liquid
Clonality: Monoclonal
Immunogen: The exact immunogen used to generate this antibody is proprietary information.
Carrier free: Yes
Clone number: EPR20261
Purification technique: Affinity purification Protein A
Concentration

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage conditions: +4°C
Appropriate long-term storage conditions: +4°C
Storage information: Do Not Freeze

Notes

What is this antibody validated in?

Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579) is a rabbit recombinant monoclonal antibody and is validated for use in Western Blot (WB), Flow Cytometry (Flow Cyt), Immunoprecipitation (IP), Immunohistochemistry (IHC-P), Immunocytochemistry/immunofluorescence (ICC/IF) in Human samples.

What is the molecular weight of LAG-3?

Anti-LAG-3 [EPR20261] - BSA and Azide free (ab227579) specifically detects a band for LAG-3 (UniProt: P18627) at a molecular weight of 57kDa.

Trial sizes available!

Test your antibody or perform pre-screening before committing to a larger quantity. Sold in 10µl. Discover our selection of trial-size antibodies.

Other related products

We have a range of other formats of antibody clone [EPR20261] also available for your convenience:
ab209236, PE - ab225484, Alexa Fluor® 488 - ab225485, Alexa Fluor® 647 - ab225486, Carrier free - ab227579, Alexa Fluor® 555 - ab280858, Carrier free - ab282638, Alexa Fluor® 594 - ab283627, ab307517, Carrier free - ab307518

Conjugation ready
Our carrier-free antibodies are typically supplied in a PBS-only formulation, purified and free of BSA, sodium azide and glycerol. This conjugation-ready format is designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, which makes them ideal for antibody labelling, functional and cell-based assays, flow-based assays (e.g. mass cytometry) and Multiplex Imaging applications.

Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with 1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

Compatibility
This product is compatible with the Maxpar^® Antibody Labeling Kit from Fluidigm, without the need for antibody preparation. Maxpar^® is a trademark of Fluidigm Canada Inc.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

LAG-3 also known as CD223 is a transmembrane protein with a mass of approximately 70 kDa. It acts as a checkpoint molecule similar to CTLA-4 and PD-1 in the immune system. LAG-3 is expressed on activated T cells natural killer cells and some dendritic cells. It interacts with its ligand MHC class II leading to inhibition of cellular proliferation and cytokine secretion an important part of immune response regulation.

Biological function summary

LAG-3 is involved in maintaining immune homeostasis and preventing autoimmunity. It is not part of a complex but works in synergy with other immune checkpoint molecules. When interacting with MHC class II LAG-3 transmits inhibitory signals that attenuate immune cell responses. Its role in immune regulation helps to balance immune activation and tolerance which is essential for preventing tissue damage while protecting against pathogens.

Pathways

The inhibitory role of LAG-3 integrates into the T cell receptor (TCR) signaling pathway and is also linked to the PD-1 signaling pathway. In TCR signaling LAG-3 negatively regulates T cell activation. LAG-3 is functionally related to proteins like PD-1 and CTLA-4 sharing similar roles in immune checkpoint pathways that are critical for maintaining immune balance.

Associated diseases and disorders

LAG-3 has links to cancer and autoimmune diseases. In cancer LAG-3 expression on tumor-infiltrating lymphocytes correlates with immune evasion by tumors. This relationship appears in various cancers including melanoma and renal cell carcinoma. In autoimmune diseases LAG-3's expression on regulatory T cells helps control conditions such as multiple sclerosis. It collaborates with CTLA-4 in modulating immune responses that prevent excessive immune activation and tissue damage associated with autoimmunity.

Product promise

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9 product images

Flow Cytometry - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)
Flow cytometric analysis of Human peripheral blood mononuclear cells treated with 1 μg/mL PHA for 3 days cells with Anti-LAG-3 antibody [EPR20261] ab209236 at 1/50 dilution (right) compared with a rabbit monoclonal IgG isotype control (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730; left). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 at 1/2000 dilution was used as the secondary antibody.
Only the CD3+ population are also positive for LAG-3. Gated on total viable cells.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-LAG-3 antibody [EPR20261] ab209236).
Flow Cytometry - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)
Flow cytometric analysis of HEK-293T (Human epithelial cell line from embryonic kidney) cells transfected with a GFP-tagged human LAG3 construct labeling LAG-3 with Anti-LAG-3 antibody [EPR20261] ab209236 at 1/500 dilution (right) compared with a rabbit monoclonal IgG isotype control (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730; left). Goat anti rabbit IgG (Alexa Fluor^® 647) Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) ab150079 at 1/2000 dilution was used as the secondary antibody.
Note: Fresh cells without fixation and permeabilization were used to perform FC testing. Only GFP positive population results in LAG3 positive staining (Q2, right panel).
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-LAG-3 antibody [EPR20261] ab209236).
Western blot - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)
This IHC data was generated using the same anti-LAG-3 antibody clone, EPR20261, in a different buffer formulation (cat# Anti-LAG-3 antibody [EPR20261] ab209236).
Primary loading control and concentration: Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) at 1/20000 dilution
Secondary loading control and concentration: Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/10000 dilution
Lanes 1-2: Merged signal (red and green). Green – Anti-LAG-3 antibody [EPR20261] ab209236 observed at 54-70 kDa. Red - loading control Anti-GAPDH antibody [6C5] - Loading Control ab8245 observed at 36 kDa.
Anti-LAG-3 antibody [EPR20261] ab209236 and Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) were incubated overnight at 4°C at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800RCW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.
The expression profile observed in Jurkat is consistent with the literature (PMID: 25108024).
Negative control: Jurkat (PMID: 25108024)
Observed MW: 54-70 kDa
All lanes: Western blot - Anti-LAG-3 antibody [EPR20261] (Anti-LAG-3 antibody [EPR20261] ab209236) at 1/500 dilution
Lane 1: HDLM-2 (Human Hodgkin lymphoma ) whole cell lysate at 20 µg
Lane 2: Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate at 20 µg
Secondary
All lanes: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/10000 dilution
Predicted band size: 57 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)
This IHC data was generated using the same anti-LAG-3 antibody clone, EPR20261, in a different buffer formulation (cat# Anti-LAG-3 antibody [EPR20261] ab209236).
Immunohistochemical analysis of paraffin-embedded human tonsil Hodgkin's lymphoma labeling LAG-3 with Anti-LAG-3 antibody [EPR20261] ab209236 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on immunocytes of the human Hodgkin's lymphoma [PMID: 11527700; PMID: 16757686].
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.
Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Immunocytochemistry/ Immunofluorescence - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)
This ICC data was generated using the same anti-LAG-3 antibody clone, EPR20261, in a different buffer formulation (cat# Anti-LAG-3 antibody [EPR20261] ab209236).
Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293T (Human epithelial cell line from embryonic kidney) cells transfected with GFP-tagged LAG3 expression construct or GFP only, labeling Lymphocyte Activation Gene 3 with Anti-LAG-3 antibody [EPR20261] ab209236 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 647) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) ab150079) secondary antibody at 1/1000 dilution (green).
Confocal image showing positive staining on HEK-293T cells transfected with a GFP-tagged LAG3 expression construct.
The nuclear counter stain is DAPI (blue). Tubulin is detected with Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 647) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) ab150079) at 1/1000 dilution.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)
Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling LAG-3 with Anti-LAG-3 antibody [EPR20261] ab209236 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Sporadic cytoplasmic staining on immunocytes of human tonsil [PMID: 11527700; PMID: 16757686].
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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-LAG-3 antibody [EPR20261] ab209236).
Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Immunoprecipitation - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)
LAG-3 was immunoprecipitated from 0.35 mg of HEK-293T (Human epithelial cell line from embryonic kidney) transfected with a GFP-tagged human LAG3 construct whole cell lysate with Anti-LAG-3 antibody [EPR20261] ab209236 at 1/30 dilution. Western blot was performed from the immunoprecipitate using Anti-LAG-3 antibody [EPR20261] ab209236 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366), was used for detection at 1/10000 dilution.
Lane 1: HEK-293T transfected with a GFP-tagged human LAG3 construct whole cell lysate 10 μg (Input).
Lane 2: Anti-LAG-3 antibody [EPR20261] ab209236 IP in HEK-293T transfected with a GFP-tagged human LAG3 construct whole cell lysate.
Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of Anti-LAG-3 antibody [EPR20261] ab209236 in HEK-293T transfected with a GFP-tagged human LAG3 construct whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 1 second.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-LAG-3 antibody [EPR20261] ab209236).
All lanes: Immunoprecipitation - Anti-LAG-3 antibody [EPR20261] (Anti-LAG-3 antibody [EPR20261] ab209236)
Predicted band size: 57 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)
Tissue Microarrays stained for "Anti-LAG-3 antibody [EPR20261]" using "Anti-LAG-3 antibody [EPR20261] ab209236"in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negative (cross mark) staining per sample type tested. The sections were pre-treated using Heat mediated antigen retrieval using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0). The sections were incubated with Anti-LAG-3 antibody [EPR20261] ab209236 at +4°C overnight followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer).
Flow Cytometry - Anti-LAG-3 antibody [EPR20261] - BSA and Azide free (ab227579)
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-LAG-3 antibody [EPR20261] ab209236).
Flow cytometry staining of human peripheral blood mononuclear cells (PBMCs) (top) or PBMCs treated with 5 μg/ml phytohaemagglutinin (PHA) for 72 hours (bottom), with Anti-LAG-3 antibody [EPR20261] ab209236 (right) or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (left). PBMCs were incubated for 30 min on ice in 1x PBS containing 10 µg/ml human IgG and 10 % normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody Anti-LAG-3 antibody [EPR20261] ab209236 or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (1x 106 in 100 µl at 5.0 μg/ml (1/402)) for 30min on ice. The cells were simultaneously stained with CD3.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min on ice

Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. Events were gated on viable cells.