Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: NDUFA9 knockout HAP1 cell lysate (20 µg)
Lane 3: WI38 cell lysate (20 µg)
Lane 4: NIH3T3 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab14713 observed at 40 kDa. Red - loading control, ab176560, observed at 52 kDa.

ab14713 was shown to specifically react with NDUFA9 in wild-type HAP1 cells. No band was observed when NDUFA9 knockout HAP1 samples were used. Wild-type and NDUFA9 knockout samples were subjected to SDS-PAGE. ab14713 and ab176560 (loading control to alpha tubulin) were diluted at 1μg/mL and 1/10,000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

ab14713 a 1/100 dilution staining NDUFA9 in Human spinal column tissue by Immunohistochemistry (Formalin/PFA-Fixed paraffin-embedded sections). Antibody was incubated with the sample for 1 hour. Sections were incubated in peroxidase-conjugated rabbit anti-mouse secondary (diluted 1/100 in 4% BSA in PBST) for 1 hour at room temperature. Sections were washed x3 in PBST and peroxidase activity was demonstrated using kit. Antigen retrieval was performed by 1 minute of pressure cooking with 1 mmol EDTA pH 8.0.

Overlay histogram showing HepG2 cells stained with ab14713 (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 (ab14713, 2µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) 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.

All lanes : Anti-NDUFA9 antibody [20C11B11B11] (ab14713) at 1 µg/ml

Lane 1 : WI38 (Human lung fibroblast cell line) Whole Cell Lysate
Lane 2 : Human testis tissue lysate - total protein (ab30257)
Lane 3 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 40 kDa
Observed band size: 36 kDa why is the actual band size different from the predicted?
Additional bands at: 58 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 20 minutes


The band observed at 36 kDa could potentially be a cleaved form of NDUFA9 due to the presence of a 35 amino acid transit peptide.

All lanes : Anti-NDUFA9 antibody [20C11B11B11] (ab14713)

Lane 1 : Isolated mitochondria from Human heart at 5 µg
Lane 2 : Isolated mitochondria from Bovine heart at 1 µg
Lane 3 : Isolated mitochondria from Rat heart at 10 µg
Lane 4 : Isolated mitochondria from Mouse Heart at 10 µg

Secondary
All lanes : Goat anti-Mouse IgG

Predicted band size: 40 kDa
Observed band size: 37 kDa why is the actual band size different from the predicted?