Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling LAG-3 with ab237719 at 1/400 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining on the human tonsil is observed. 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.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

LAG-3 was immunoprecipitated from 0.35 mg of LAG-3-GFP transfected HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen)  whole cell lysate with ab237719 at 1/20 dilution. Western blot was performed from the immunoprecipitate using ab237719 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used as secondary antibody at 1/5000 dilution.

Lane 1: LAG-3-GFP transfected HEK-293T whole cell lysate 10 μg (Input).
Lane 2: ab237719 IP in LAG-3-GFP transfected HEK-293T whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab237719 in LAG-3-GFP transfected HEK-293T whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 30 seconds.

Flow cytometric analysis of 2% paraformaldehyde-fixed, 0.1% Tween-20 permeabilized human peripheral blood mononuclear cells ((PBMC) (treated with 10μg/ml PHA for 48 hours) cell line labeling LAG-3 with ab237719 at 1/250 dilution (Right) compared with Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (Left).

Goat Anti-Rabbit IgG Fc (Alexa Fluor^® 488) preadsorbed (ab150097), at 1/5000 dilution was used as the secondary antibody.

Cells were surface stained with anti-CD3 conjugated to Alexa Fluor^® 647. Then fixed with 2% PFA for 10min followed by intracellular staining rabbit IgG (Left) or ab237719 (Right).

All lanes : Anti-LAG-3 antibody [CAL26] (ab237719) at 1/1000 dilution

Lane 1 : Untransfected HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 2 : LAG-3-GFP overexpression HEK-293T whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 57 kDa
Observed band size: 16,70 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking and dilution buffer: 5% NFDM/TBST.

This antibody also detects a 16kDa cleaved fragment. (PMID: 15557174).

All lanes : Anti-LAG-3 antibody [CAL26] (ab237719) at 1/1000 dilution

Lane 1 : Human peripheral blood mononuclear cell (PBMC), whole cell lysate
Lane 2 : Human peripheral blood mononuclear cell (PBMC) treated with 10µg/ml PHA for 48 hours, whole cell lysate
Lane 3 : Human lymphoma lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 57 kDa
Observed band size: 16,70 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking and dilution buffer: 5% NFDM/TBST.

This antibody also detects a 16kDa cleaved fragment. (PMID: 15557174).

 

Formalin-fixed, paraffin-embedded human tonsil tissue stained for LAG-3 using ab237719 at 0.3 μg/ml in immunohistochemical analysis.