Anti-LEF1 抗体 [EPR2029Y]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LEF1 antibody [EPR2029Y] (AB137872)

ab137872 staining LEF1 in paraffin embedded human melanoma tissue by Immunohistochemistry. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.  Samples were incubated with primary antibody at 1/2000 dilution for 30 mins at room temperature. A ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Hematoxylin was used as a counterstain. The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-LEF1 antibody [EPR2029Y] (AB137872)

Flow cytometry overlay histogram showing left Jurkat positive cells and right negative HeLa stained with ab137872 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab137872) (1x 106 in 100μl at 0.2μg/ml (1/11500 dilution)) for 30min at 22°C.The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed was incubated at 1/4000 for 30min at 22°CIsotype control antibody (black line) was Recombinant Rabbit IgG monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.This antibody gave a positive signal in Jurkat Fixed with 4% formaldehyde (10 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LEF1 antibody [EPR2029Y] (AB137872)

ab137872 staining LEF1 in paraffin embedded human thymona tissue by Immunohistochemistry. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.  Samples were incubated with primary antibody at 1/2000 dilution for 30 mins at room temperature. A ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Hematoxylin was used as a counterstain. The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LEF1 antibody [EPR2029Y] (AB137872)

ab137872 staining LEF1 in paraffin embedded human melanoma tissue by Immunohistochemistry. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0 Epitope Retrieval Solution2) for 20 mins.  Samples were incubated with primary antibody at 1/2000 dilution for 30 mins at room temperature. A ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Hematoxylin was used as a counterstain. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

• WB

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Western blot - Anti-LEF1 antibody [EPR2029Y] (AB137872)

Blocking/Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-LEF1 antibody [EPR2029Y] (ab137872) at 1/1000 dilution

All lanes:

Human fetal thymus lysate at 10 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 44 kDa

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• WB

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Western blot - Anti-LEF1 antibody [EPR2029Y] (AB137872)

Blocking/Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-LEF1 antibody [EPR2029Y] (ab137872) at 1/10000 dilution

All lanes:

Jurkat (Human T cell leukemia cell line from peripheral blood) cell lysate at 10 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 44 kDa

false

• WB

Unknown

Western blot - Anti-LEF1 antibody [EPR2029Y] (AB137872)

Blocking/Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-LEF1 antibody [EPR2029Y] (ab137872) at 1/2000 dilution

All lanes:

Rat thymus tissue lysate at 20 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 44 kDa

false

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-LEF1 antibody [EPR2029Y] (AB137872)

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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-LEF1 antibody [EPR2029Y] (AB137872)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LEF1 antibody [EPR2029Y] (AB137872)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LEF1 antibody [EPR2029Y] (AB137872)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LEF1 antibody [EPR2029Y] (AB137872)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LEF1 antibody [EPR2029Y] (AB137872)

Tissue Microarrays stained for Anti-LEF1 antibody [EPR2029Y] using ab137872 in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negaive (cross mark) staining per sample type tested. The sections were incubated with ab137872 for 30 mins at room temperature used at 1 : 2000 dilution (1.05 μg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB secondary antibody (ab209101). Counterstain was Hematoxylin. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins. The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LEF1 antibody [EPR2029Y] (AB137872)

Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling LEF1 with ab137872 at a concentration of 0.5 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 32 mins at 100°C with ULTRA cell conditioning solution (CC1, pH 8.5). ab137872 anti LEF1 antibody [EPR2029Y] was incubated at 37°C for 16 mins. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LEF1 antibody [EPR2029Y] (AB137872)

ab137872 staining LEF1 in paraffin embedded mouse spleen tissue by Immunohistochemistry. Antigen retrieval was performed 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).

• IP

Lab

Immunoprecipitation - Anti-LEF1 antibody [EPR2029Y] (AB137872)

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

All lanes:

Immunoprecipitation - Anti-LEF1 antibody [EPR2029Y] (ab137872)

Predicted band size: 44 kDa

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Exposure time: 3min

• IP

Lab

Immunoprecipitation - Anti-LEF1 antibody [EPR2029Y] (AB137872)

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

All lanes:

Immunoprecipitation - Anti-LEF1 antibody [EPR2029Y] (ab137872)

Predicted band size: 44 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-LEF1 antibody [EPR2029Y] (AB137872)

Different batches of ab137872 were tested on Jurkat (Human T cell leukemia T lymphocyte) lysate at 1.1 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 25-57 kDa.

All lanes:

Western blot - Anti-LEF1 antibody [EPR2029Y] (ab137872)

Predicted band size: 44 kDa

false

• WB

Lab

Western blot - 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.

All lanes:

Western blot - Anti-LEF1 antibody [EPR2029Y] (ab137872) at 1/1000 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='/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

• WB

Lab

Western blot - Anti-LEF1 antibody [EPR2029Y] (AB137872)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-LEF1 antibody [EPR2029Y] (ab137872) at 1/1000 dilution

All lanes:

His-tagged mouse LEF-1 recombinant protein (aa1-397) at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 44 kDa

Observed band size: 44 kDa

false

Exposure time: 1s