- PA5-143906 - Invitrogen Antibodies WAPL antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of WAPL using WAPL antibody (Product # PA5-143906). Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 µg of sample under reducing conditions. Lane 1: human Hela whole cell lysates; Lane 2: human 293T whole cell lysates; Lane 3: human HepG2 whole cell lysates; Lane 4: rat L6 whole cell lysates; Lane 5: mouse C2C12 whole cell lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-WAPL/FOE antigen affinity purified polyclonal antibody (Product # PA5-143906) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0. 1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5,000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit with Tanon 5200 system. A specific band was detected for WAPL/FOE at approximately 180 kDa. The expected band size for WAPL/FOE is at 133 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunocytochemistry analysis of WAPL in T-47D cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum. Samples were then incubated in WAPL Polyclonal antibody (Product # PA5-143906) using a dilution of 5 μg/mL. DyLight®594 Conjugated Goat Anti-Rabbit IgG was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of WAPL in paraffin-embedded section of mouse colon tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. Samples were incubated with WAPL Polyclonal antibody (Product # PA5-143906) using a dilution of 2 μg/mL overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit with DAB as the chromogen.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of WAPL in paraffin-embedded section of human lymphoma tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. Samples were incubated with WAPL Polyclonal antibody (Product # PA5-143906) using a dilution of 2 μg/mL overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit with DAB as the chromogen.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of WAPL in paraffin-embedded section of human stomach cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. Samples were incubated with WAPL Polyclonal antibody (Product # PA5-143906) using a dilution of 2 μg/mL overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit with DAB as the chromogen.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of WAPL in paraffin-embedded section of human tonsil tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. Samples were incubated with WAPL Polyclonal antibody (Product # PA5-143906) using a dilution of 2 μg/mL overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit with DAB as the chromogen.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of WAPL in A431 cells using WAPL Polyclonal Antibody (Product # PA5-143906), shown in overlay histogram (blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum, and incubated with the primary antibody (1 μg/1x10^6 cells) for 30 min at 20°C. DyLight 488 conjugated goat anti-rabbit IgG (5-10 µg/1x10^6 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 µg/1x10^6) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

PA5-143906