Anti-LAG-3 antibody [EPR20261]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [EPR20261] (AB209236)

Tissue Microarrays stained for "Anti-LAG-3 antibody [EPR20261]" using "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 ab93684 (Tris/EDTA buffer, pH 9.0). The sections were incubated with ab209236 at +4°C overnight followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer).

• Flow Cyt

Lab

Flow Cytometry - 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 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 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.

• IHC-P

Supplier Data

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.

• Flow Cyt

Lab

Flow Cytometry - Anti-LAG-3 antibody [EPR20261] (AB209236)

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.

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-LAG-3 antibody [EPR20261] (AB209236)

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).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - 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 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.

• IHC-P

Supplier Data

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.

• IP

Supplier Data

Immunoprecipitation - Anti-LAG-3 antibody [EPR20261] (AB209236)

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.

All lanes:

Immunoprecipitation - Anti-LAG-3 antibody [EPR20261] (ab209236)

Predicted band size: 57 kDa

false

• WB

Unknown

Western blot - Anti-LAG-3 antibody [EPR20261] (AB209236)

Primary 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

All lanes:

Western blot - 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 (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 57 kDa

false

• WB

Supplier Data

Western blot - Anti-LAG-3 antibody [EPR20261] (AB209236)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-LAG-3 antibody [EPR20261] (ab209236) at 1/1000 dilution

All lanes:

Human LAG3 Fc chimera recombinant protein (aa23-450) at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 57 kDa

Observed band size: 90 kDa

false

Exposure time: 1s

• WB

Lab

Western blot - Anti-LAG-3 antibody [EPR20261] (AB209236)

Blocking/Diluting buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-LAG-3 antibody [EPR20261] (ab209236) at 1/5000 dilution

Lane 1:

293T (Human embryonic kidney epithelial cell) transfected with an empty vector (vector control), containing a GFP tag, whole cell lysate at 15 µg

Lane 2:

293T transfected with LAG3 expression vector containing a GFP tag, whole cell lysate at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 57 kDa

false

Exposure time: 5s