EIF4A3 Antibody / Eukaryotic initiation factor 4A-III (FY13354)

Immunofluorescent staining of EIF4A3 using anti-EIF4A3 antibody (green) and anti-Beta Tubulin antibody (red). EIF4A3 was detected in an immunocytochemical section of human A549 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-EIF4A3 antibody and mouse anti-Beta Tubulin antibody overnight at 4oC. DyLight 488 Conjugated Goat Anti-Rabbit IgG and Cy3 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.

Western blot analysis of EIF4A3 using anti-EIF4A3 antibody. Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human whole cell lysates, Lane 2: human 293T whole cell lysates, Lane 3: human Hela whole cell lysates, Lane 4: human MCF-7 whole cell lysates, Lane 5: rat liver tissue lysates, Lane 6: rat lung tissue lysates, Lane 7: mouse liver tissue lysates, Lane 8: mouse lung 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-EIF4A3 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 specific band was detected for EIF4A3 at approximately 47 kDa. The expected molecular weight of EIF4A3 is ~47 kDa.

Immunohistochemical staining of EIF4A3 using anti-EIF4A3 antibody. EIF4A3 was detected in a paraffin-embedded section of human prostate cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 ug/ml rabbit anti-EIF4A3 antibody overnight at 4oC. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37oC. The tissue section was developed using an HRP secondary and DAB substrate.

Immunohistochemical staining of EIF4A3 using anti-EIF4A3 antibody. EIF4A3 was detected in a paraffin-embedded section of human cervical cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 ug/ml rabbit anti-EIF4A3 antibody overnight at 4oC. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37oC. The tissue section was developed using an HRP secondary and DAB substrate.

Immunoprecipitating EIF4A3 in 293T whole cell lysate. Western blot analysis of EIF4A3 using anti-EIF4A3 antibody. Lane 1: 293T whole cell lysates (30ug), Lane 2: Rabbit control IgG instead of anti-EIF4A3 antibody in 293T whole cell lysate, Lane 3: anti-EIF4A3 antibody (2ug) + 293T whole cell lysate (500ug). After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-EIF4A3 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 specific band was detected for EIF4A3 at approximately 47 kDa. The expected molecular weight of EIF4A3 is at 47 kDa.

Flow Cytometry analysis of 293T cells using anti-EIF4A3 antibody. Overlay histogram showing 293T 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-EIF4A3 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.