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

• Catalog Number: orb738423
• Category: Antibodies
• Description: Anti-ATP5F1, 2, 3/ATP5MC1, 2, 3 Antibody (monoclonal, 12E9). Tested in Flow Cytometry, WB applications. This antibody reacts with Human, Monkey, Mouse, Rat.
• Species/Host: Mouse
• Clonality: Monoclonal
• Clone Number: 12E9
• Tested applications: FC, WB
• Reactivity: Human, Monkey, Mouse, Rat
• Isotype: Mouse IgG2b
• Immunogen: E.coli-derived human ATP5G1,2,3/ATP5MC1,2,3 recombinant protein (Position: D62-L113).
• Antibody Type: Primary Antibody
• Concentration: Adding 0.2 ml of distilled water will yield a concentration of 500 μg/ml.
• Form/Appearance: Lyophilized
• Conjugation: Unconjugated
• MW: 10-14 kDa
• UniProt ID: P05496
• Storage: Maintain refrigerated at 2-8°C for up to 2 weeks. For long term storage store at -20°C in small aliquots to prevent freeze-thaw cycles.
• Alternative names: ATP synthase D chain mitochondrial antibody; ATP s
• Note: For research use only
• Application notes: Western blot, 0.25-0.5μg/ml, Human, Mouse, Rat, Monkey Flow Cytometry (Fixed), 1-3μg/1x106 cells, Human, Mouse, Rat. Add 0.2ml of distilled water will yield a concentration of 500ug/ml
• Expiration Date: 12 months from date of receipt.

Flow Cytometry analysis of HEPA1-6 cells using anti-ATP5F1, 2, 3/ATP5MC1, 2, 3 antibody. Overlay histogram showing HEPA1-6 cells (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 (1 µg/1x10^6 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (5-10 µg/1x10^6 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1 µg/1x10^6) 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. Overlay histogram showing HEPG2 cells (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 (1 µg/1x10^6 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (5-10 µg/1x10^6 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1 µg/1x10^6) 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. Overlay histogram showing RH35 cells (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 (1 µg/1x10^6 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (5-10 µg/1x10^6 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1 µg/1x10^6) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

Western blot analysis of ATP5F1, 2, 3/ATP5MC1, 2, 3 using anti-ATP5F1, 2, 3/ATP5MC1, 2, 3 antibody. 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 50 ug 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 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 mouse anti-ATP5F1, 2, 3/ATP5MC1, 2, 3 antigen affinity purified monoclonal antibody 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 with Tanon 5200 system. A specific band was detected for ATP5F1, 2, 3/ATP5MC1, 2, 3 at approximately 10-14 KD. The expected band size for ATP5F1, 2, 3/ATP5MC1, 2, 3 is at 10-14 KD.