Recombinant Anti-LEF1 antibody [EP2030Y] (ab53293)

False colour image of Western blot: Anti-LEF1 antibody [EP2030Y] staining at 1/5000 dilution, shown in black; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab53293 was shown to bind specifically to LEF1. A band was observed at 40/53 kDa in wild-type Jurkat cell lysates with no signal observed at this size in Lef1 knockout cell line ab274898 (knockout cell lysate ab274956). To generate this image, wild-type and Lef1 knockout Jurkat cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% milk in TBS-0.1 % Tween^® 20 (TBS-T) 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 before development with Optiblot (ECL reagent ab133456) and imaged with 8 minutes exposure time. Secondary antibodies used were HRP conjugated Goat anti-Rabbit (H+L) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

This antibody recognizes all 7 LEF1 isoforms with the MW ranging from 23 to 44 KD based on immunogen blast.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human tonsil tissue sections labeling LEF1 with purified ab53293 at 1/50 dilution (1.94 µg/ml). Heat mediated antigen retrieval was performed using heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunocytochemistry/ Immunofluorescence analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling LEF1 with purified ab53293 at 1/50 dilution (2.0 µg/ml). Cells were fixed in 100% Methanol. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Intracellular Flow Cytometry analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling LEF1 with purified ab53293 at 1/20 dilution (10µg/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue). 

ab53293 (unpurified) staining LEF1 in RAMOS (human Burkitt's lymphoma) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/500. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody at a dilution of 1/1000. DAPI was used as a nuclear counterstain and the negative control was PBS only.

Paraffin-embedded human colon adenocarcinoma tissue stained for LEF1 using ab53293 (unpurified) at 1/50 dilution in immunohistochemical analysis.

Intracellular flow cytometric analysis of permeabilized Jurkat cells using ab53293 (unpurified) (red) or a rabbit IgG (negative) (green).