NUP98 Antibody / Nucleoporin 98 (FY12859)

Immunofluorescent staining of NUP98 using anti-NUP98 antibody (red). NUP98 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-NUP98 antibody overnight at 4oC. Cy3 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 NUP98 using anti-NUP98 antibody. Lane 1: human Caco-2 whole cell lysates, Lane 2: human Hela whole cell lysates, Lane 3: human K562 whole cell lysates, Lane 4: monkey COS-7 whole cell lysates, Lane 5: rat NRK whole cell lysates, Lane 6: rat PC-12 whole cell lysates, Lane 7: mouse RAW264.7 whole cell lysates, Lane 8: mouse NIH/3T3 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-NUP98 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. NUP98 western blot across human and rodent cell lines shows a predominant band just above the 100 kDa marker with a lighter band just below 100 kDa. The paired bands are consistent with differentially modified forms of NUP98, which commonly runs slightly above its predicted 98 kDa due to O-GlcNAc and phosphorylation.

Flow Cytometry analysis of K562 cells using anti-NUP98 antibody. Overlay histogram showing K562 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-NUP98 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.