Anti-Annexin A3/ANXA3 Antibody Picoband®

Western blot analysis of ANXA3 using anti-ANXA3 antibody (PB9420).
Electrophoresis was performed on a 10% 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 A431 whole cell lysates,
Lane 2: human Caco-2 whole cell lysates,
Lane 3: human SiHa whole cell lysates,
Lane 4: human Hela whole cell lysates,
Lane 5: rat lung tissue lysates,
Lane 6: rat kidney tissue lysates,
Lane 7: mouse lung tissue lysates,
Lane 8: mouse kidney tissue 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-ANXA3 antigen affinity purified polyclonal antibody (PB9420) 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 ANXA3 at approximately 36 kDa. The expected band size for ANXA3 is at 36 kDa.

IHC analysis of ANXA3 using anti-ANXA3 antibody (PB9420).
ANXA3 was detected in a paraffin-embedded section of human gastric 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-ANXA3 Antibody (PB9420) 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.

IF analysis of ANXA3 using antiANXA3 antibody (PB9420).
ANXA3 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-ANXA3 Antibody (PB9420) overnight at 4°C. Fluoro488 Conjugated Goat Anti-Rabbit IgG (BA1127) was used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Immunoprecipitating ANXA3 in A431 whole cell lysate.
Western blot analysis of ANXA3 using anti-ANXA3 antibody (PB9420);
Lane 1: A431 whole cell lysates (30ug);
Lane 2: Rabbit control IgG instead of anti-ANXA3 antibody in A431 whole cell lysate;
Lane 3: anti-ANXA3 antibody (2μg) + A431 whole cell lysate (500μg).
After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-ANXA3 antigen affinity purified polyclonal antibody (PB9420) 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 ANXA3 at approximately 36 kDa. The expected band size for ANXA3 is at 36 kDa.

Flow Cytometry analysis of Hela cells using anti-ANXA3 antibody (PB9420).
Overlay histogram showing Hela cells stained with PB9420 (Blue line). The cells were fixed with 4% paraformaldehyde and blocked with 10% normal goat serum. And then incubated with rabbit anti-ANXA3 Antibody (PB9420, 1 μg/1x10⁶ cells) for 30 min at 20°C. Fluoro488 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.