Anti-Carbonic Anhydrase 9/CA9 Antibody Picoband®

Figure 1. Western blot analysis of Carbonic Anhydrase 9/CA9 using anti-Carbonic Anhydrase 9/CA9 antibody (A01083-3).
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 30 ug of sample under reducing conditions.
Lane 1: human U-87MG whole cell lysates,
Lane 2: human 293T 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-Carbonic Anhydrase 9/CA9 antigen affinity purified polyclonal antibody (Catalog # A01083-3) 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 Carbonic Anhydrase 9/CA9 at approximately 54 kDa. The expected band size for Carbonic Anhydrase 9/CA9 is at 50 kDa.

Figure 2. IHC analysis of Carbonic Anhydrase 9/CA9 using anti-Carbonic Anhydrase 9/CA9 antibody (A01083-3).
Carbonic Anhydrase 9/CA9 was detected in a paraffin-embedded section of human colonic adenocarcinoma tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-Carbonic Anhydrase 9/CA9 Antibody (A01083-3) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

Figure 3. IHC analysis of Carbonic Anhydrase 9/CA9 using anti-Carbonic Anhydrase 9/CA9 antibody (A01083-3).
Carbonic Anhydrase 9/CA9 was detected in a paraffin-embedded section of human renal adenocarcinoma tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-Carbonic Anhydrase 9/CA9 Antibody (A01083-3) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

Figure 4. IHC analysis of Carbonic Anhydrase 9/CA9 using anti-Carbonic Anhydrase 9/CA9 antibody (A01083-3).
Carbonic Anhydrase 9/CA9 was detected in a paraffin-embedded section of mouse stomach tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-Carbonic Anhydrase 9/CA9 Antibody (A01083-3) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

Figure 5. Flow Cytometry analysis of U87 cells using anti-Carbonic Anhydrase 9/CA9 antibody (A01083-3).
Overlay histogram showing U87 cells stained with A01083-3 (Blue line). The cells were fixed with 4% paraformaldehyde and blocked with 10% normal goat serum. And then incubated with rabbit anti-Carbonic Anhydrase 9/CA9 Antibody (A01083-3, 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.

Figure 6. Flow Cytometry analysis of RAW264.7 cells using anti-Carbonic Anhydrase 9/CA9 antibody (A01083-3).
Overlay histogram showing RAW264.7 cells stained with A01083-3 (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-Carbonic Anhydrase 9/CA9 Antibody (A01083-3, 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.