Flow Cytometry analysis of U20S cells using anti-14-3-3 SIGMA antibody. Overlay histogram showing U20S cells (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 then incubated with rabbit anti-14-3-3 SIGMA Antibody (1 µg/1x10 6 cells) for 30 min at 20C. DyLight488 conjugated goat anti-rabbit IgG (5-10 µg/1x10 6 cells) was used as secondary antibody for 30 minutes at 20C. Isotype control antibody (Green line) was rabbit IgG (1 µg/1x10 6) used under the same conditions. Unlabelled sample (Red line) was also used as a control.
WB analysis of 14-3-3 sigma using anti-14-3-3 sigma antibody
IF analysis of 14-3-3 sigma using anti-14-3-3 sigma antibody. 14-3-3 sigma was detected in paraffin-embedded section of A431 cell. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 µg/mL rabbit anti-cortactin Antibody overnight at 4C. DyLight488 Conjugated Goat Anti-Rabbit IgG was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37C. Visualize using a fluorescence microscope and filter sets appropriate for the label used.
IF analysis of 14-3-3 sigma using anti-14-3-3 sigma antibody. 14-3-3 sigma was detected in paraffin-embedded section of A549 cell. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 µg/mL rabbit anti-cortactin Antibody overnight at 4C. DyLight488 Conjugated Goat Anti-Rabbit IgG was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37C. Visualize using a fluorescence microscope and filter sets appropriate for the label used.
IHC analysis of 14-3-3 sigma using anti-14-3-3 sigma antibody. 14-3-3 sigma was detected in paraffin-embedded section of human rectal cancer tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1 µg/ml rabbit anti-14-3-3 sigma Antibody overnight at 4C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen.
Western blot analysis of 14-3-3 sigma using anti-14-3-3 sigma antibody. 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 ug of sample under reducing conditions. Lane 1: human A431 whole cell lysates, Lane 2: human Hela whole cell lysates, Lane 3: human MCF-7 whole cell lysates, Lane 4: human Hacat whole cell lysates, Lane 5: rat skin tissue lysates, Lane 6: mouse Hepa1-6 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-14-3-3 sigma antigen affinity purified polyclonal antibody at 0.5 µg/mL overnight at 4C, 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 1:5000 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 14-3-3 sigma at approximately 28 kDa. The expected band size for 14-3-3 sigma is at 28 kDa.
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