NRCAM Antibody / Neuronal cell adhesion molecule (FY12537)

Flow Cytometry analysis of Neuro2a cells using anti-NRCAM antibody. Overlay histogram showing Neuro2a 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-NRCAM 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 NRCAM using anti-NRCAM antibody. Electrophoresis was performed on a 8% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: rat brain tissue lysates, Lane 2: rat PC-12 whole cell lysates, Lane 3: mouse C6 whole cell lysates, Lane 4: mouse brain tissue lysates, Lane 5: mouse SP2/0 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-NRCAM 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. NRCAM (~144 kDa predicted) was detected as a doublet around ~136 kDa, consistent with the known differential glycosylation and proteolytic processing that produce multiple mature NrCAM species.