SMAP2 Antibody / Small ArfGAP 2 (FY13074)

Flow Cytometry analysis of THP-1 cells using anti-SMAP2 antibody. Overlay histogram showing THP-1 cells stained with (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-SMAP2 antibody (1 ug/million cells) for 30 min at 20oC. DyLight 488 conjugated goat anti-rabbit IgG (5-10 ug/million cells) was used as secondary antibody for 30 minutes at 20oC. Isotype control antibody (Green line) was rabbit IgG (1 ug/million cells) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

Western blot analysis of SMAP2 using anti-SMAP2 antibody. Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human THP-1 whole cell lysates, Lane 2: human K562 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-SMAP2 antibody at 0.5 ug/ml overnight at 4oC, 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 was developed using an ECL Plus Western Blotting Substrate. A major band is detected at approximately 42 kDa, slightly below the predicted molecular weight of 47 kDa. This difference is consistent with previous reports and reflects the faster electrophoretic migration of the mature SMAP2 protein, which lacks extensive post-translational modifications and may undergo minor N-terminal processing. The observed band corresponds to the full-length SMAP2 protein.

Immunoprecipitating (IP) SMAP2 in K562 whole cell lysate. Western blot analysis of SMAP2 using anti-SMAP2 antibody; Lane 1: K562 whole cell lysates (30ug); Lane 2: Rabbit control IgG instead of anti-SMAP2 antibody in K562 whole cell lysate; Lane 3: anti-SMAP2 antibody (2ug) + K562 whole cell lysate (500ug). After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-SMAP2 antibody at a dilution of 0.5 ug/ml and probed with a mouse anti-rabbit IgG-HRP secondary antibody. The signal is developed using ECL Plus Western Blotting Substrate. A major band is detected at approximately 42 kDa, slightly below the predicted molecular weight of 47 kDa. This difference is consistent with previous reports and reflects the faster electrophoretic migration of the mature SMAP2 protein, which lacks extensive post-translational modifications and may undergo minor N-terminal processing. The observed band corresponds to the full-length SMAP2 protein.