SH2B1 Antibody / SH2B adapter protein 1 (FY12892)

Flow Cytometry analysis of SH-SY5Y cells using anti-SH2B1 antibody. Overlay histogram showing SH-SY5Y 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-SH2B1 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 SH2B1 using anti-SH2B1 antibody. Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human 293T whole cell lysates, Lane 2: human Hela whole cell lysates, Lane 3: human SH-SY5Y whole cell lysates, Lane 4: rat brain tissue lysates, Lane 5: 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-SH2B1 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 light ~70 kDa and stronger ~85 kDa band are observed in human lysates, consistent with expression of shorter and full-length isoforms. Mouse and rat brain samples display a 90-100 kDa doublet and higher 140-150 kDa doublet, likely reflecting phosphorylated and complexed forms of SH2B1 previously reported in neuronal tissue.