Anti-ATP5F1,2,3/ATP5MC1,2,3 Picoband® Antibody (monoclonal, 12E9)

Western blot analysis of ATP5F1,2,3/ATP5MC1,2,3 using anti-ATP5F1,2,3/ATP5MC1,2,3 antibody (M09735).
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 HL-60 whole cell lysates,
Lane 2: monkey COS-7 whole cell lysates,
Lane 3: rat kidney tissue lysates,
Lane 4: rat heart tissue lysates,
Lane 5: mouse heart tissue 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 mouse anti-ATP5F1,2,3/ATP5MC1,2,3 antigen affinity purified monoclonal antibody (Catalog # M09735) 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-mouse 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 # EK1001) with Tanon 5200 system. A specific band was detected for ATP5F1,2,3/ATP5MC1,2,3 at approximately 10-14KD. The expected band size for ATP5F1,2,3/ATP5MC1,2,3 is at 10-14KD.

Flow Cytometry analysis of HEPA1-6 cells using anti-ATP5F1,2,3/ATP5MC1,2,3 antibody (M09735).
Overlay histogram showing HEPA1-6 cells stained with M09735 (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 mouse anti- ATP5F1,2,3/ATP5MC1,2,3 Antibody (M09735, 1μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (BA1126, 5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse 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.

Flow Cytometry analysis of HEPG2 cells using anti-ATP5F1,2,3/ATP5MC1,2,3 antibody (M09735).
Overlay histogram showing HEPG2 cells stained with M09735 (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 mouse anti- ATP5F1,2,3/ATP5MC1,2,3 Antibody (M09735, 1μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (BA1126, 5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse 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.

Flow Cytometry analysis of RH35 cells using anti-ATP5F1,2,3/ATP5MC1,2,3 antibody (M09735).
Overlay histogram showing RH35 cells stained with M09735 (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 mouse anti- ATP5F1,2,3/ATP5MC1,2,3 Antibody (M09735, 1μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (BA1126, 5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse 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.