Anti-LAG-3 antibody [EPR20261] - Low endotoxin, Azide free

• Flow Cyt

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Flow Cytometry - Anti-LAG-3 antibody [EPR20261] - Low endotoxin, Azide free (AB282638)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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 LAG-3. Gated on total viable cells.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-LAG-3 antibody [EPR20261] - Low endotoxin, Azide free (AB282638)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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 LAG-3 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.

• Flow Cyt

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Flow Cytometry - Anti-LAG-3 antibody [EPR20261] - Low endotoxin, Azide free (AB282638)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab209236).

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 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).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [EPR20261] - Low endotoxin, Azide free (AB282638)

This IHC data was generated using the same anti-LAG-3 antibody clone, EPR20261, in a different buffer formulation (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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [EPR20261] - Low endotoxin, Azide free (AB282638)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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.

• WB

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Western blot - Anti-LAG-3 antibody [EPR20261] - Low endotoxin, Azide free (AB282638)

This IHC data was generated using the same anti-LAG-3 antibody clone, EPR20261, in a different buffer formulation (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.

All lanes:

Western blot - Anti-LAG-3 antibody [EPR20261] - Low endotoxin, Azide free (ab282638)

Predicted band size: 57 kDa

false

• WB

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Western blot - Anti-LAG-3 antibody [EPR20261] - Low endotoxin, Azide free (AB282638)

This IHC data was generated using the same anti-LAG-3 antibody clone, EPR20261, in a different buffer formulation (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] - Low endotoxin, Azide free (ab282638) 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='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 57 kDa

false