All lanes : Anti-ATPase Inhibitory Factor 1/IF1 antibody [5E2D7] (ab110277) at 1 µg/ml

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : ATPase Inhibitory Factor 1 / IF1 knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 12 kDa

Lanes 1 - 3: Merged signal (red and green). Green - ab110277 observed at 12 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab110277 was shown to recognize ATPase Inhibitory Factor 1 / IF1 in wild-type HAP1 cells as signal was lost at the expected MW in ATPase Inhibitory Factor 1 / IF1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and ATPase Inhibitory Factor 1 / IF1 knockout samples were subjected to SDS-PAGE. Ab110277 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

ICC/IF image of ab110277 stained HepG2 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab110277, 10µg/ml) overnight at +4°C. The secondary antibody (green) was ab96879, DyLight® 488 goat anti-mouse IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM

All lanes : Anti-ATPase Inhibitory Factor 1/IF1 antibody [5E2D7] (ab110277) at 1 µg/ml

Lane 1 : Human heart mitochondria at 10 µg
Lane 2 : Bovine heart mitochondria at 4 µg
Lane 3 : Rat heart mitochondria at 10 µg
Lane 4 : Mouse heart mitochondria at 10 µg

Predicted band size: 12 kDa

Overlay histogram showing HepG2 cells stained with ab110277 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab110277, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab91353) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.