STAM2 Antibody / Signal transducing adaptor molecule 2 (FY13090)

Immunofluorescent staining of STAM2 using anti-STAM2 antibody (red) and anti-Beta Tubulin antibody (green). STAM2 was detected in immunocytochemical section of PC-3 cell. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 ug/ml rabbit anti-STAM2 antibody and mouse anti-Beta Tubulin antibody overnight at 4oC. Cy3 Conjugated Goat Anti-Rabbit IgG and DyLight 488 Conjugated Goat Anti-Mouse IgG were used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37oC. Visualize using a fluorescence microscope and filter sets appropriate for the label used. U2OS cells were stained with STAM2 antibody (red) and Beta-tubulin antibody (green). STAM2 displays cytoplasmic punctate staining, concentrated in the perinuclear region consistent with its association with early/sorting endosomes. The vesicular STAM2 signal partially aligns with the microtubule network, reflecting its role in endosome trafficking along microtubule tracks.

Western blot analysis of STAM2 using anti-STAM2 antibody. Lane 1: human HepG2 whole cell lysates, Lane 2: rat liver tissue lysates, Lane 3: rat brain tissue lysates, Lane 4: mouse brain 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-STAM2 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 enhanced chemiluminescent. Although STAM2 is ~58-60 kDa by sequence, the antibody detects bands at ~65-70 kDa, consistent with the known phosphorylation-dependent upward shift and presence of two isoforms (STAM2A/B). In brain tissue lysates, STAM2 appears as a doublet, likely reflecting different phosphorylation states and/or isoform usage.

Flow Cytometry analysis of Hela cells using anti-STAM2 antibody. Overlay histogram showing Hela 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-STAM2 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 (Red line) was also used as a control.