Anti-Angiogenin/ANG Antibody Picoband™

Figure 1. Western blot analysis of Angiogenin/ANG using anti-Angiogenin/ANG antibody (A00146).
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 hepatocellular carcinoma tumor tissue (HCCT) 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-Angiogenin/ANG antigen affinity purified polyclonal antibody (Catalog # A00146) 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 Angiogenin/ANG at approximately 17 kDa. The expected band size for Angiogenin/ANG is at 17 kDa.

Figure 2. IHC analysis of Angiogenin/ANG using anti-Angiogenin/ANG antibody (A00146).
Angiogenin/ANG was detected in a paraffin-embedded section of human hepatocellular carcinoma 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-Angiogenin/ANG Antibody (A00146) 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 Angiogenin/ANG using anti-Angiogenin/ANG antibody (A00146).
Angiogenin/ANG was detected in a paraffin-embedded section of human lung cancer 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-Angiogenin/ANG Antibody (A00146) 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. IF analysis of Angiogenin/ANG using anti-Angiogenin/ANG antibody (A00146) and anti-Beta Tubulin antibody (M01857-3).
Angiogenin/ANG was detected in immunocytochemical section of U2OS 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-Angiogenin/ANG Antibody (A00146) and mouse anti-Beta Tubulin antibody (M01857-3) overnight at 4°C. Cy3 Conjugated Goat Anti-Rabbit IgG (BA1032) and FITC Conjugated Goat Anti-Mouse IgG (BA1101) 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.

Figure 5. Flow Cytometry analysis of HepG2 cells using anti-Angiogenin/ANG antibody (A00146).
Overlay histogram showing HepG2 cells stained with A00146 (Blue line). The cells were fixed with 4% paraformaldehyde and blocked with 10% normal goat serum. And then incubated with rabbit anti-Angiogenin/ANG Antibody (A00146, 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 (Red line) was also used as a control.

Figure 6. Sandwich ELISA - Recombinant human ANG protein standard curve.
Use in combination with reagents from Human ANG ELISA Kit EZ-Set (DIY Antibody Pairs) (EZ0305).