EPS8 Antibody / Epidermal growth factor receptor pathway substrate 8 (FY13369)

Immunofluorescent staining of EPS8 using anti-EPS8 antibody (green). EPS8 was detected in an immunocytochemical section of cells. 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-EPS8 antibody overnight at 4oC. DyLight 488 Conjugated Goat Anti-Rabbit IgG was used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37oC. The section was counterstained with DAPI nuclear stain (blue). Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Western blot analysis of EPS8 using anti-EPS8 antibody. Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human RT4 whole cell lysates, Lane 2: human whole cell lysates, Lane 3: human HepG2 whole cell lysates, Lane 4: human whole cell lysates, Lane 5: mouse C6 whole cell lysates, Lane 6: 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-EPS8 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 molecular weight of EPS8 is ~92 kDa.

Immunoprecipitating EPS8 in whole cell lysate. Western blot analysis of EPS8 using anti-EPS8 antibody. Lane 1: whole cell lysates (30ug), Lane 2: Rabbit control IgG instead of anti-EPS8 antibody in whole cell lysate, Lane 3: anti-EPS8 antibody (2ug) + whole cell lysate (500ug). After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-EPS8 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 EPS8 at approximately 97 kDa. The expected molecular weight of EPS8 is at 92 kDa.

Flow Cytometry analysis of cells using anti-EPS8 antibody. Overlay histogram showing cells stained with (Blue line). The cells were fixed with 4% paraformaldehyde and blocked with 10% normal goat serum. And then incubated with rabbit anti-EPS8 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.