NAP1L4 Antibody / Nucleosome assembly protein 1-like 4 (FY13033)

Flow Cytometry analysis of 293T cells using anti-NAP1L4 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-NAP1L4 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.

Western blot analysis of NAP1L4 using anti-NAP1L4 antibody. Lane 1: human Hela whole cell lysates, Lane 2: human 293T whole cell lysates, Lane 3: human whole cell lysates, Lane 4: human HepG2 whole cell lysates, Lane 5: rat testis tissue lysates, Lane 6: mouse testis 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-NAP1L4 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 major doublet is detected at approximately 65 kDa, higher than the predicted 43 kDa molecular weight. This slower migration is characteristic of NAP1 family histone chaperones and reflects acidic domain composition and post-translational modifications, including phosphorylation and acetylation, which generate distinct electrophoretic species of NAP1L4 on SDS-PAGE.