Anti-Smac/Diablo Antibody Picoband®

Western blot analysis of Smac/Diablo using anti-Smac/Diablo antibody (PB9860).
Electrophoresis was performed on a 12 SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human 293T whole cell lysates,
Lane 2: human Hela whole cell lysates,
Lane 3: human HepG2 whole cell lysates,
Lane 4: humna Jurkat whole cell lysates,
Lane 5: rat testis tissue lysates,
Lane 6: mouse testis tissue 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-Smac/Diablo antigen affinity purified polyclonal antibody (Catalog # PB9860) 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 1:5000 for 1.5 hour at RT. The signal is developed using an ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for Smac/Diablo at approximately 20 kDa. The expected band size for Smac/Diablo is at 27 kDa.

IHC analysis of Smac/Diablo using anti-Smac/Diablo antibody (PB9860).
Smac/Diablo was detected in a paraffin-embedded section of human rectal 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. The tissue section was then incubated with 2 μg/ml rabbit anti-Smac/Diablo Antibody (PB9860) 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 (Catalog # SV0002) with DAB as the chromogen.

IF analysis of Smac/Diablo using anti-Smac/Diablo antibody (PB9860) and anti-Tubulin Alpha antibody (M03989-3).
Smac/Diablo was detected in immunocytochemical section of U2OS cell. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 μg/mL rabbit anti-Smac/Diablo Antibody (PB9860) and mouse anti-Tubulin Alpha antibody (M03989-3) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) and Cy3 Conjugated Goat Anti-Mouse IgG (BA1031) were used as secondary antibody at 1:500 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.

Immunoprecipitating (IP) Smac/Diablo in HepG2 whole cell lysate.
Western blot analysis of Smac/Diablo using anti-Smac/Diablo antibody (PB9860);
Lane 1: HepG2 whole cell lysates (30ug);
Lane 2: Rabbit control IgG instead of anti-Smac/Diablo antibody in HepG2 whole cell lysate;
Lane 3: anti-Smac/Diablo antibody (2μg) + HepG2 whole cell lysate (500μg).
After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-Smac/Diablo antigen affinity purified polyclonal antibody (PB9860) at a dilution of 0.5 μg/mL and probed with a goat anti-rabbit IgG-HRP secondary antibody (Catalog # BA1054). The signal is developed using ECL Plus Western Blotting Substrate (Catalog # AR1196-200). A specific band was detected for Smac/Diablo at approximately 20 kDa. The expected band size for Smac/Diablo is at 27 kDa.

Flow Cytometry analysis of 293T cells using anti-Smac/Diablo antibody (PB9860).
Overlay histogram showing 293T cells stained with PB9860 (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-Smac/Diablo Antibody (PB9860, 1 μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10 μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.