Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: NDUFS2 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: Jurkat whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab110249 observed at 48 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab110249 was shown to specifically react with NDUFS2 in wild-type HAP1 cells whilst signal was lost in NDUFS2 knockout cells. Wild-type and NDUFS2 knockout samples were subjected to SDS-PAGE. ab110249 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 0.25 µg/ml and 1/20,000 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 1/20,000 dilution for 1 hour at room temperature before imaging.

Overlay histogram showing HepG2 cells stained with ab110249 (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 (ab110249, 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) (ab150113) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

All lanes : Anti-NDUFS2 antibody [7A12BE5AD5] (ab110249) at 0.25 µg/ml

Lane 1 : Molecular Weight Markers
Lane 2 : Isolated mitochondria from Human liver
Lane 3 : Isolated mitochondria from Bovine heart
Lane 4 : Isolated mitochondria from H9C2 cells (Rat cardiomyocyte)
Lane 5 : Isolated mitochondria from MEF cells (Mouse embryo fibroblast)
Lane 6 : Isolated mitochondria from HepG2

Predicted band size: 53 kDa



Complex I-subunit NDUFS2 is detected in the Human samples with Human specific ab110249, while Complex I-subunit NDUFB8 is detected in all samples (Human, Mouse and Rat) with an anti-NDUFB8.

Mitochondrial localization of Complex I subunit NDUFS2 visualized by Immunocytochemistry in HDFn cultured cells (normal Human dermal fibroblasts, neonatal), using ab110249 at 5 µg/ml.