Complex I was immunopurified from mitochondria isolated from human heart (HHM), cow/bovine heart (BHM), mouse heart (MHM) and mouse brain (MBM).  The lanes were stained with Coomassie Brilliant Blue R. Bands were excised from the gel and proteolytically digested for mass spectrometry analysis. For the immuno-isolation, 50 μg of mAb (18G12BC2 ab109798) was bound to 5 μl of swollen protein G agarose beads according to protocol described here.

Immunocytochemistry image of ab109798 stained fibroblasts cells. The cells were paraformaldehyde fixed (4%, 20 minutes) and Triton X-100 permeabilized (0.1%, 15 minutes). The cells were incubated with the antibody (ab109798, 1 µg/mL) for 2 hours at room temperature or over night at 4°C. The secondary antibody was (green) Alexa Fluor® 4884 goat anti-mouse IgG (H+L) at a 1/1000 dilution for 1 hour. 10% Goat serum was used as the blocking agent for all blocking steps. The target protein locates to the mitochondria.

HL-60 cells were stained with 1 µg/mL Complex I antibody ab109798 (blue) or an equal amount of an isotype control antibody (red) and analyzed by flow cytometry.

Overlay histogram showing HepG2 cells stained with ab109798 (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. The cells were then incubated with the antibody (ab109798, 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 IgG2b [PLPV219] (ab91366, 2µg/1x10⁶ cells ) used under the same conditions. Acquisition of >5,000 events was performed.