NLE1 Antibody / Notchless protein homolog 1 (FY12416)

Flow Cytometry analysis of 293T cells using anti-NLE1 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-NLE1 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 NLE1 using anti-NLE1 antibody. Lane 1: human PC-3 whole cell lysates, Lane 2: human 293T whole cell lysates, Lane 3: human whole cell lysates, Lane 4: human U251 whole cell lysates, Lane 5: rat brain tissue lysates, Lane 6: rat ovary tissue lysates, Lane 7: mouse brain tissue lysates, Lane 8: mouse ovary 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-NLE1 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. NLE1 (~51 kDa predicted) was detected at ~45-48 kDa, occasionally as a doublet, consistent with the processed and phosphorylated forms reported in ribosome assembly studies.