Recombinant Anti-LAG-3 antibody [EPR20261] (ab209236)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR20261] to LAG-3
- Suitable for: WB, IHC-P, ICC/IF, Flow Cyt, IP
- Reacts with: Human
Overview
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Product name
Anti-LAG-3 antibody [EPR20261]
See all LAG-3 primary antibodies -
Description
Rabbit monoclonal [EPR20261] to LAG-3 -
Host species
Rabbit -
Tested Applications & Species
Application Species Flow Cyt HumanICC/IF HumanIHC-P HumanIP HumanWB Human -
Immunogen
Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: HDLM-2 cells and Human LAG3 Fc chimera recombinant protein (aa23-450). IHC-P: Human tonsil and Hodgkin’s lymphoma tissues. ICC/IF: HEK-293T cells transfected with a GFP-tagged LAG3 expression construct. Flow Cyt: HEK-293T transfected with a GFP-tagged human LAG3 construct. IP: HEK-293T transfected with a GFP-tagged human LAG3 construct whole cell lysate.
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General notes
This product was previously labelled as Lymphocyte Activation Gene 3.
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol, 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR20261 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Conjugation kits
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Isotype control
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab209236 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Tested applications are guaranteed to work and covered by our Abpromise guarantee.
Predicted to work for this combination of applications and species but not guaranteed.
Does not work for this combination of applications and species.
Application | Species |
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Flow Cyt |
Human
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ICC/IF |
Human
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IHC-P |
Human
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IP |
Human
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WB |
Human
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Application | Abreviews | Notes |
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WB |
1/500. Detects a band of approximately 70 kDa (predicted molecular weight: 54 kDa).
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IHC-P | (1) |
1/1000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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. |
ICC/IF |
1/100.
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Flow Cyt |
1/500.
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IP |
1/30.
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Notes |
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WB
1/500. Detects a band of approximately 70 kDa (predicted molecular weight: 54 kDa). |
IHC-P
1/1000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. 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. |
ICC/IF
1/100. |
Flow Cyt
1/500. |
IP
1/30. |
Target
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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
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Database links
- Entrez Gene: 3902 Human
- Omim: 153337 Human
- SwissProt: P18627 Human
- Unigene: 409523 Human
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Alternative names
- CD223 antibody
- CD223 antigen antibody
- FDC protein antibody
see all
Images
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All lanes : Anti-LAG-3 antibody [EPR20261] (ab209236) at 1/500 dilution
Lane 1 : HDLM-2 (Human Hodgkin lymphoma ) whole cell lysate
Lane 2 : Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution (Goat anti-Rabbit IgG H&L (IRDye® 800RCW) preadsorbed)
Predicted band size: 54 kDaPrimary loading control and concentration: 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 (ab216776) at 1/10000 dilution
Lanes 1-2: Merged signal (red and green). Green – ab209236 observed at 54-70 kDa. Red - loading control ab8245 observed at 36 kDa.
ab209236 and 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 (ab216773) and 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
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [EPR20261] (ab209236)
Immunohistochemical analysis of paraffin-embedded human tonsil Hodgkin’s lymphoma labeling LAG-3 with 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.
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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 LAG-3 with ab209236 at 1/100 dilution, followed by Goat anti-rabbit IgG (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 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) (ab150079) at 1/1000 dilution.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [EPR20261] (ab209236)
Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling LAG-3 with 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.
Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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Flow cytometric analysis of Human peripheral blood mononuclear cells treated with 1 μg/mL PHA for 3 days cells with ab209236 at 1/50 dilution (right) compared with a rabbit monoclonal IgG isotype control (ab172730; left). ab150077 at 1/2000 dilution was used as the secondary antibody.
Only the CD3+ population are also positive for LAG3. Gated on total viable cells.
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Flow cytometric analysis of HEK-293T (Human epithelial cell line from embryonic kidney) cells transfected with a GFP-tagged human LAG-3 construct labeling LAG-3 with ab209236 at 1/500 dilution (right) compared with a rabbit monoclonal IgG isotype control (ab172730; left). Goat anti rabbit IgG (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).
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Anti-LAG-3 antibody [EPR20261] (ab209236) at 1/1000 dilution + Human LAG3 Fc chimera recombinant protein (aa23-450) at 0.01 µg
Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 54 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?
Exposure time: 1 secondBlocking/Dilution buffer: 5% NFDM/TBST.
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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 ab209236 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab209236 at 1/1000 dilution. 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: ab209236 IP in HEK-293T transfected with a GFP-tagged human LAG3 construct whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of 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.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
Certificate of Compliance
References (1)
ab209236 has been referenced in 1 publication.
- Wang H et al. Altered expression of TIM-3, LAG-3, IDO, PD-L1, and CTLA-4 during nimotuzumab therapy correlates with responses and prognosis of oral squamous cell carcinoma patients. J Oral Pathol Med 48:669-676 (2019). PubMed: 31132187