NYAP2 Antibody / Neuronal tyrosine-phosphorylated phosphoinositide-3-kinase adapter 2 (FY13249)

Flow Cytometry analysis of human SIHA cells using anti-NYAP2 antibody. Overlay histogram showing SIHA 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-NYAP2 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 NYAP2 using anti-NYAP2 antibody. Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human Hela whole cell lysates, Lane 2: human SIHA whole cell lysates, Lane 3: human SH-SY5Y whole cell lysates, Lane 4: human U2OS 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-NYAP2 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. Western blot analysis of NYAP2 in HeLa, SiHa, SH-SY5Y, and U-251 cell lysates using an anti NYAP2 antibody. A predominant band is detected at an approximately 80-85 kDa in all samples, running slightly above the predicted ~71 kDa size of NYAP2 but consistent with the higher apparent molecular weight reported for this heavily phosphorylated adaptor protein.