Anti-CD73/Nt5e Antibody Picoband®

Figure 1. Western blot analysis of CD73/Nt5e using anti-CD73/Nt5e antibody (A02120-2).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions.
Lane 1: rat PC-12 whole cell lysates,
Lane 2: rat C6 whole cell lysates,
Lane 3: rat RH35 whole cell lysates,
Lane 4: mouse thymus tissue lysates.
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA 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-CD73/Nt5e antigen affinity purified polyclonal antibody (Catalog # A02120-2) 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 Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for CD73/Nt5e at approximately 63KD. The expected band size for CD73/Nt5e is at 63KD.

Figure 2. IHC analysis of CD73/Nt5e using anti-CD73/Nt5e antibody (A02120-2).
CD73/Nt5e was detected in paraffin-embedded section of mouse spleen tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-CD73/Nt5e Antibody (A02120-2) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1022) with DAB as the chromogen.

Figure 3. Flow Cytometry analysis of mouse spleen tissues using anti-CD73/Nt5e antibody (A02120-2).
Overlay histogram showing mouse spleen tissue stained with A02120-2 (Blue line). To facilitate intracellular staining, tissues were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The tissues were blocked with 10% normal goat serum. And then incubated with rabbit anti-CD73/Nt5e Antibody (A02120-2, 1μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

Figure 4. Flow Cytometry analysis of mouse spleen tissue using anti-CD73/Nt5e antibody (A02120-2).
Overlay histogram showing mouse spleen tissue stained with A02120-2 (Blue line).The tissue was blocked with 10% normal goat serum. And then incubated with rabbit anti-CD73/Nt5e Antibody (A02120-2, 1μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1μg/1x10⁶) used under the same conditions. Unlabelled sample (Red line) was also used as a control.