STAT5A/B Antibody / Signal transducer and activator of transcription 5 (FY12778)

Immunofluorescent staining of STAT5A/B using anti-STAT5A/B antibody (red) and anti-Beta Tubulin antibody (green). STAT5A/B was detected in immunocytochemical section of HELA 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-STAT5A/B 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.

Western blot analysis of STAT5A/B using anti-STAT5A/B antibody. Lane 1: human Hela whole cell lysates, Lane 2: human HepG2 whole cell lysates, Lane 3: human K562 whole cell lysates, Lane 4: human whole cell lysates, Lane 5: rat PC-12 whole cell lysates, Lane 6: mouse thymus 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-STAT5A/B 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. A doublet is observed between ~85-90 kDa, corresponding to STAT5A and STAT5B isoforms (predicted ~91 kDa). The minor migration difference reflects known phosphorylation- and isoform-dependent mobility shifts.

Flow Cytometry analysis of cells using anti-STAT5A/B antibody. Overlay histogram showing 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-STAT5A/B 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.