Anti-NCAM2 Antibody Picoband®

Western blot analysis of NCAM2 using anti-NCAM2 antibody (A06583-2).
Electrophoresis was performed on a 8% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human MCF-7 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-NCAM2 antigen affinity purified polyclonal antibody (A06583-2) at 0.5 μg/mL overnight at 4°C, 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 is developed using an ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for NCAM2 at approximately 140 kDa. The expected band size for NCAM2 is at 93 kDa.

IF analysis of NCAM2 using anti-NCAM2 antibody (A06583-2) and anti-Beta Tubulin antibody (M01857-3).
NCAM2 was detected in an immunocytochemical section of Hela cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 μg/mL rabbit anti-NCAM2 Antibody (A06583-2) and mouse anti-Beta Tubulin antibody (M01857-3) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) and Cy3 Conjugated Goat Anti-Mouse IgG (BA1031) were used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37°C. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Immunoprecipitating NCAM2 in MCF-7 whole cell lysate.
Western blot analysis of NCAM2 using anti-NCAM2 antibody (A06583-2).
Lane 1: MCF-7 whole cell lysates (30ug),
Lane 2: Rabbit control IgG instead of anti-NCAM2 antibody in MCF-7 whole cell lysate,
Lane 3: anti-NCAM2 antibody (2μg) + MCF-7 whole cell lysate (500μg).
After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-NCAM2 antigen affinity purified polyclonal antibody (A06583-2) at a dilution of 0.5 μg/mL and probed with a goat anti-rabbit IgG-HRP secondary antibody (Catalog # BA1054). The signal is developed using ECL Plus Western Blotting Substrate (Catalog # AR1197). A specific band was detected for NCAM2 at approximately 140 kDa. The expected band size for NCAM2 is at 93 kDa.

Flow Cytometry analysis of THP-1 cells using anti-NCAM2 antibody (A06583-2).
Overlay histogram showing THP-1 cells stained with A06583-2 (Blue line). The cells were fixed with 4% paraformaldehyde and blocked with 10% normal goat serum. And then incubated with rabbit anti-NCAM2 Antibody (A06583-2, 1 μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10 μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.