Anti-Cytochrome P450 3A4/CYP3A4 Antibody Picoband®

Western blot analysis of Cytochrome P450 3A4/CYP3A4 using anti-Cytochrome P450 3A4/CYP3A4 antibody (A00339-2).
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 30 ug of sample under reducing conditions.
Lane 1: human SH-SY5Y whole cell lysates,
Lane 2: human HL-60 whole cell lysates,
Lane 3: human K562 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-Cytochrome P450 3A4/CYP3A4 antigen affinity purified polyclonal antibody (Catalog # A00339-2) 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 Cytochrome P450 3A4/CYP3A4 at approximately 57 kDa. The expected band size for Cytochrome P450 3A4/CYP3A4 is at 57 kDa.

Western blot analysis of Cytochrome P450 3A4/CYP3A4 using anti-Cytochrome P450 3A4/CYP3A4 antibody (A00339-2).
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 30 ug of sample under reducing conditions.
Lane 1-2: Human keratinocytes isolated from skin.
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-Cytochrome P450 3A4/CYP3A4 antigen affinity purified polyclonal antibody (Catalog # A00339-2) 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 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit with ChemiDoc MP system. A specific band was detected for Cytochrome P450 3A4/CYP3A4 at approximately 57 kDa. The expected band size for Cytochrome P450 3A4/CYP3A4 is at 57 kDa.

Flow Cytometry analysis of HL-60 cells using anti-Cytochrome P450 3A4/CYP3A4 antibody (A00339-2).
Overlay histogram showing HL-60 cells stained with A00339-2 (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-Cytochrome P450 3A4/CYP3A4 Antibody (A00339-2, 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.

Flow Cytometry analysis of U87 cells using anti-Cytochrome P450 3A4/CYP3A4 antibody (A00339-2).
Overlay histogram showing U87 cells stained with A00339-2 (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-Cytochrome P450 3A4/CYP3A4 Antibody (A00339-2, 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.