NFIA Antibody / Nuclear factor 1 A-type (FY12453)

Flow Cytometry analysis of JK cells using anti-NFIA antibody. Overlay histogram showing JK 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-NFIA 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 NFIA using anti-NFIA antibody. Lane 1: human whole cell lysates, Lane 2: human Hela whole cell lysates, Lane 3: human Jurkat whole cell lysates, Lane 4: human MCF-7 whole cell lysates, Lane 5: rat RH35 whole cell lysates, Lane 6: mouse L929 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-NFIA 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. NFIA (~55 kDa predicted) was detected at ~70 kDa, consistent with phosphorylation- and acetylation-dependent mobility shifts reported for the active nuclear form of this transcription factor.