NPW Antibody / Neuropeptide W (FY13053)

Flow Cytometry analysis of HepG2 cells using anti-NPW antibody. Overlay histogram showing HepG2 cells stained with (Blue line). The cells were fixed with 4% paraformaldehyde and blocked with 10% normal goat serum. And then incubated with rabbit anti-NPW 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 NPW using anti-NPW antibody. Electrophoresis was performed on a 12% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human PC-3 whole cell lysates, Lane 2: human HepG2 whole cell lysates, Lane 3: human K562 whole cell lysates, Lane 4: mouse lung tissue lysates, Lane 5: mouse stomach 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-NPW 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 an ECL Plus Western Blotting Substrate. A single band is detected at approximately 14 kDa, lower than the predicted 18 kDa for the unprocessed precursor. This apparent size corresponds to the processed prohormone form of NPW following removal of its N-terminal signal peptide and proteolytic maturation into smaller secretory intermediates. Similar migration (~13-15 kDa) has been reported for endogenous NPW in mammalian brain and peripheral tissues.