Anti-NHP2 Antibody Picoband®

Western blot analysis of NHP2 using anti-NHP2 antibody (A04887-1).
Electrophoresis was performed on a 12% 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 SH-SY5Y whole cell lysates,
Lane 2: human Caco-2 whole cell lysates,
Lane 3: human Hela 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-NHP2 antigen affinity purified polyclonal antibody (A04887-1) 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 (Catalog # BA1054) 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 NHP2 at approximately 20 kDa. The expected band size for NHP2 is at 17 kDa.

IF analysis of NHP2 using anti-NHP2 antibody (A05665-1) and anti-Beta Tubulin antibody (M01857-3).
NHP2 was detected in immunocytochemical section of A549 cell. 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-NHP2 Antibody (A05665-1) 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 NHP2 in SH-SY5Y whole cell lysate.
Western blot analysis of NHP2 using anti-NHP2 antibody (A05665-1);
Lane 1: SH-SY5Y whole cell lysates (30ug);
Lane 2: Rabbit control IgG instead of anti-NHP2 antibody in SH-SY5Y whole cell lysate;
Lane 3: anti-NHP2 antibody (2μg) + SH-SY5Y whole cell lysate (500μg).
After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-NHP2 antigen affinity purified polyclonal antibody (A05665-1) 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 # AR1196-200). A specific band was detected for NHP2 at approximately 20 kDa. The expected band size for NHP2 is at 17 kDa.

Flow Cytometry analysis of CACO-2 cells using anti-NHP2 antibody (A05665-1).
Overlay histogram showing CACO-2 cells stained with A05665-1 (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-NHP2 Antibody (A05665-1, 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.