Recombinant Anti-LAG-3 antibody [CAL26] (ab237719)

False colour image of Western blot: Anti-LAG-3 antibody [CAL26] 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, ab237719 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.

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).

Blocking and dilution buffer: 5% NFDM/TBST.

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

This blot was developed using a higher sensitivity ECL substrate.

Blocking and dilution buffer: 5% NFDM/TBST.

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

This blot was developed using a higher sensitivity ECL substrate.

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