Anti-LEF1 antibody [EP2030Y] - BSA and Azide free

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-LEF1 antibody [EP2030Y] - BSA and Azide free (AB227562)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab53293).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LEF1 antibody [EP2030Y] - BSA and Azide free (AB227562)

This IHC data was generated using the same anti-LEF1 antibody clone, EP2030Y, in a different buffer formulation (cat# ab53293).

ab53293 (unpurified) (1 : 50) staining human LEF1 in human colon adenocarcinoma tissue by immunohistochemistry using paraffin embedded tissue.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LEF1 antibody [EP2030Y] - BSA and Azide free (AB227562)

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 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab53293).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-LEF1 antibody [EP2030Y] - BSA and Azide free (AB227562)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab53293).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-LEF1 antibody [EP2030Y] - BSA and Azide free (AB227562)

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 Fluor^® 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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab53293).

• WB

Lab

Western blot - Anti-LEF1 antibody [EP2030Y] - BSA and Azide free (AB227562)

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 data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab53293).

All lanes:

Western blot - Anti-LEF1 antibody [EP2030Y] (<a href='/en-us/products/primary-antibodies/lef1-antibody-ep2030y-ab53293'>ab53293</a>) at 1/5000 dilution

Lane 1:

Wild-type Jurkat cell lysate at 40 µg

Lane 2:

Lef1 knockout Jurkat cell lysate at 40 µg

Lane 2:

Western blot - Human LEF1 knockout Jurkat cell line (<a href='/en-us/products/cell-lines/human-lef1-knockout-jurkat-cell-line-ab274898'>ab274898</a>)

Lane 3:

Jurkat cell lysate at 20 µg

Predicted band size: 44 kDa

Observed band size: 40 kDa

false

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-LEF1 antibody [EP2030Y] - BSA and Azide free (AB227562)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab53293).