Anti-LAG-3 antibody [CAL25]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [CAL25] (AB237718)

Formalin-fixed, paraffin-embedded human tonsil tissue stained for LAG-3 using ab237718 at 0.3 μg/ml in immunohistochemical analysis.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAG-3 antibody [CAL25] (AB237718)

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).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-LAG-3 antibody [CAL25] (AB237718)

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).

• IP

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Immunoprecipitation - Anti-LAG-3 antibody [CAL25] (AB237718)

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)

All lanes:

Immunoprecipitation - Anti-LAG-3 antibody [CAL25] (ab237718)

Predicted band size: 57 kDa

Observed band size: 16 kDa,70 kDa

false

• WB

Lab

Western blot - Anti-LAG-3 antibody [CAL25] (AB237718)

False colour image of Western blot : Anti-LAG-3 antibody [CAL25] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab237718 was shown to bind specifically to LAG-3. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-LAG-3 antibody [CAL25] (ab237718) at 1/1000 dilution

Lane 1:

HDLM-2 cell lysate at 20 µg

Lane 2:

Jurkat cell lysate at 20 µg

Predicted band size: 57 kDa

Observed band size: 57 kDa,70 kDa

false

• WB

Supplier Data

Western blot - Anti-LAG-3 antibody [CAL25] (AB237718)

Blocking buffer and concentration : 5% NFDM/TBST

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

All lanes:

Western blot - Anti-LAG-3 antibody [CAL25] (ab237718) at 1/1000 dilution

Lane 1:

Human peripheral blood mononuclear cell (PBMC) whole cell lysate at 20 µg

Lane 2:

Human peripheral blood mononuclear cell (PBMC) treated with 10μg/ml PHA for 48h whole cell lysate at 20 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: 16 kDa,70 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-LAG-3 antibody [CAL25] (AB237718)

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

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

All lanes:

Western blot - Anti-LAG-3 antibody [CAL25] (ab237718) at 1/1000 dilution

Lane 1:

HEK-293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

HEK-293T transfected with GFP-tagged LAG-3 expression vector whole cell lysate at 20 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: 16 kDa,70 kDa

false

Exposure time: 3min