Recombinant Anti-LEF1 antibody [EPR2029Y] (ab137872)

False colour image of Western blot: Anti-LEF1 antibody [EPR2029Y] staining at 1/1000 dilution, shown in black; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab137872 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 4 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.

Blocking/Dilution buffer: 5% NFDM/TBST

Immunohistochemical staining of paraffin embedded human tonsil with purified ab137872 at a working dilution of 1/500. The secondary antibody used is ab97051, an HRP-conjugated goat anti-rabbit IgG (H+L), at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Immunofluorescence staining of Jurkat (Human T cell leukemia cell line from peripheral blood) cells with purified ab137872 at a working dilution of 1 in 500, counter-stained with DAPI. Tubulin was stained with mouse anti-tubulin at a dilution of 1/1000 (ab7291) and Alexa Fluor^® 594 goat anti-mouse at a dilution of 1/500 (ab150120) . The secondary antibody was ab150077 Alexa Fluor^® 488 goat anti rabbit, used at a dilution of 1 in 500. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100.

The negative controls are shown in the bottom middle and right hand panels - for the first negative control, purified ab137872 was used at a dilution of 1/200 followed by an Alexa Fluor^® 555 goat anti-mouse antibody at a dilution of 1/500 and for the second negative control mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab15007) were used.

Intracellular flow cytometric analysis of Jurkat cell line (human T cell leukemia T lymphocyte) fixed with 4% paraformaldehyde and permeabilized with 90% methanol labeling LEF1 with ab137872 at 1/600 dilution (red). This is compared with a Rabbit monoclonal IgG (ab172730) - Isotype control (black) and a unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti-rabbit IgG (Alexa Fluor^®488) was used as the secondary antibody. 

Lane 1 (input): Jurkat (human T cell leukemia T lymphocyte) whole cell lysate, 10 μg
Lane 2 (+): Jurkat whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of  ab137872 in Jurkat  whole cell lysate

ab137872 immunoprecipitating LEF1 in Jurkat whole cell lysate. For western blotting, primary antibody used was ab137872 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

Blocking and diluting buffer: 5% NFDM/TBST

Exposure time: 3 minutes

Immunohistochemical staining of paraffin embedded rat spleen with purified ab137872 at a working dilution of 1/500. The secondary antibody used is ab97051, an HRP-conjugated goat anti-rabbit IgG (H+L), at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Immunohistochemical staining of paraffin-embedded human thymus with purified ab137872 at a working dilution of 1/500. The secondary antibody used is ab97051, an HRP-conjugated goat anti-rabbit IgG (H+L), at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

ab137872 staining LEF1 in paraffin embedded mouse spleen tissue by Immunohistochemistry. Antigen retrieval was by heat mediation using ab93684 (Tris/EDTA buffer, ph 9).  Samples were incubated with primary antibody at 1/2000 dilution. A ready to use Goat Anti-rabbit IgG H&L (HRP) was used as the secondary antibody. Hematoxylin was used as a counterstain. Nuclear staining on T cells in periarterial lymphatic sheath of mouse spleen is observed (PMID: 21685909).

Blocking/Dilution buffer: 5% NFDM/TBST

Blocking/Dilution buffer: 5% NFDM/TBST

Blocking and diluting buffer: 5% NFDM/TBST

Lane 1 (input): Rat thymus lysate, 10μg
Lane 2 (+): Rat thymus lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of  ab137872 in rat thymus lysate

ab137872 immunoprecipitating LEF1 in rat thymus lysate. For western blotting, primary antibody used was ab137872 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

Blocking and diluting buffer: 5% NFDM/TBST

Exposure time: 3 minutes