Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling LAG-3 using ab237718 at 1/800 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining on the human tonsil is observed. The section was incubated with ab237718 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP). 

LAG-3 was immunoprecipitated from 0.35 mg HEK-293T (human embryonic kidney epithelial cell) transfected with GFP-tagged LAG-3 expression vector whole cell lysate using ab237718 at 1/30 dilution. Western blot was performed on the immunoprecipitate using ab237718 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used as the secondary antibody at 1/5000 dilution.

Lane 1: HEK-293T  transfected with GFP-tagged LAG-3 expression vector whole cell lysate 10 µg (input).
Lane 2: ab237718 IP in HEK-293T GFP-tagged LAG-3 expression vector whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab237718 in 293T transfected with GFP-tagged LAG-3 expression vector whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 30 seconds.

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

Flow cytometric analysis of 2% paraformaldehyde-fixed 0.1% Tween-20 permeabilized Human peripheral blood mononuclear cells (PBMC) treated with 10μg/ml PHA for 48h labeling LAG-3 with ab237718 at 1/50 dilution (Right) compared to a Rabbit monoclonal IgG (ab172730, Left). Goat anti rabbit IgG (Alexa Fluor® 488, ab150097) was used as the secondary at 1/5000 dilution.

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 ab237718 (Right).

All lanes : Anti-LAG-3 antibody [CAL25] (ab237718) at 1/1000 dilution

Lane 1 : HEK-293T (human embryonic kidney epithelial cell) whole cell lysate
Lane 2 : HEK-293T transfected with GFP-tagged LAG-3 expression vector 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: 70, 16 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/diluting buffer and concentration: 5% NFDM/TBST

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

All lanes : Anti-LAG-3 antibody [CAL25] (ab237718) 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 48h 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: 70, 16 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking buffer and concentration: 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 ab237718 at 0.3 μg/ml in immunohistochemical analysis.