Anti-BCRP/ABCG2 Antibody Picoband®

Western blot analysis of BCRP/ABCG2 using anti-BCRP/ABCG2 antibody (A00457-2).
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: human A549 whole cell lysates,
Lane 2: human MDA-MB-453 whole cell lysates,
Lane 3: human HepG2 whole cell lysates,
Lane 4: human Caco-2 whole cell lysates,
Lane 5: human PC-3 whole cell lysates,
Lane 6: rat liver tissue lysates,
Lane 7: rat kidney tissue lysates,
Lane 8: mouse HEPA1-6 whole cell 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-BCRP/ABCG2 antigen affinity purified polyclonal antibody (Catalog # A00457-2) 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 1:10000 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 BCRP/ABCG2 at approximately 65-80KD. The expected band size for BCRP/ABCG2 is at 72KD.

Western blot analysis of BCRP using anti-BCRP antibody (A00457-2).
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 μg of sample under reducing conditions.
Lane 1: huamn MDA-MB-231 whole cell lysates,
Lane 2: human MDA-MB-231 whole cell lysates,
Lane 3: human HCC1937 whole cell lysates,
Lane 4: human HCC1937 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-BCRP antigen affinity purified polyclonal antibody (A00457-2) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween-20 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 BCRP at approximately 72 kDa. The expected band size for BCRP is at 72 kDa.

IHC analysis of BCRP/ABCG2 using anti-BCRP/ABCG2 antibody (A00457-2).
BCRP/ABCG2 was detected in paraffin-embedded section of human mammary cancer 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 2μg/ml rabbit anti-BCRP/ABCG2 Antibody (A00457-2) 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.

IHC analysis of BCRP/ABCG2 using anti-BCRP/ABCG2 antibody (A00457-2).
BCRP/ABCG2 was detected in paraffin-embedded section of mouse liver 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 2μg/ml rabbit anti-BCRP/ABCG2 Antibody (A00457-2) 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.

Flow Cytometry analysis of SiHa cells using anti-BCRP/ABCG2 antibody (A00457-2).
Overlay histogram showing SiHa cells stained with A00457-2 (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-BCRP/ABCG2 Antibody (A00457-2,1μg/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.