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

All lanes : 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
Lane 2 : 293T transfected with LAG3 expression vector containing a GFP tag, whole cell lysate

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 54 kDa


Exposure time: 5 seconds

Blocking/Diluting buffer: 5% NFDM/TBST.

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 kDa

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

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.

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.

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 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 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).

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 second

Blocking/Dilution buffer: 5% NFDM/TBST.

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.