EFS Antibody / Embryonal Fyn-associated substrate (FY12187)

Flow Cytometry analysis of SH-SY5Y cells using anti-EFS 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-EFS 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 EFS using anti-EFS antibody. Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human SH-SY5Y whole cell lysates, Lane 2: human RT4 whole cell lysates, Lane 3: human SIHA whole cell lysates, Lane 4: rat ovary tissue lysates, Lane 5: rat PC-12 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-EFS 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. The expected band size for EFS is at 59 kDa but can be observed at 80-90 kDa due to extensive phosphorylation.

Immunoprecipitating (IP) EFS in SiHa whole cell lysate. Western blot analysis of EFS using anti-EFS antibody; Lane 1: SiHa whole cell lysates (30ug); Lane 2: Rabbit control IgG instead of anti-EFS antibody in SiHa whole cell lysate; Lane 3: anti-EFS antibody (2ug) + SiHa whole cell lysate (500ug). After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-EFS antibody at a dilution of 0.5 ug/ml and probed with a goat anti-rabbit IgG-HRP secondary antibody. The signal is developed using ECL Plus Western Blotting Substrate. A specific band was detected for EFS at approximately 85 kDa. The expected band size for EFS is at 59 kDa.