Anti-SDHA antibody [2E3GC12FB2AE2] (ab14715)

This data was developed using the same antibody clone in a different format (HRP conjugated) (ab198493).

Lanes 1 - 4: Merged signal (red and green). Green - ab198493 observed at 72 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab198493 was shown to recognize in wild-type HEK-293 cells as signal was lost at the expected MW in SDHA knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and SDHA knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. Ab198493 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 1/5000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 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.

250 μm-thick formalin-fixed human cerebellum section were passively cleared using PACT and immunofluorescently labelled to identify mitochondrial mass (porin (ab14734, 1/100) and SDHA (ab14715, 1/100), 647 nm) and complex I subunits within the mitochondrial respiratory chain (NDUFB8 (ab110242, 1/100) and NDUFA13 (ab110240, 1/100); 546 nm) in conjunction with a neuronal marker (NF-H; 488 nm) in control 1. Scale: 100 μm.

Mitochondrial localization of complex II visualized by immunocytochemistry using ab14715. Cultured human embryonic lung-derived fibroblasts (strain MRC5) were fixed, permeabilized and then labeled with ab14715 (0.2 µg/ml) followed by an AlexaFluor^® 488-conjugated-goat-anti-mouse IgG2a isotype specific secondary antibody (2 µg/ml).

Human skeletal muscle immunohistochemistry using ab14715. Fixed frozen tissue sections from a patient with a single large deletion of the mtDNA were used. All muscle fibers exhibit complex II immunoreactivity, consistent with the nuclear DNA-encoded expression pattern of this and all other subunits of complex II.

ICC/IF image of ab14715 stained human HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 0.1% PBS-tween diluted 1%BSA (OR 10% goat serum OR 0.3M glycine) for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab14715, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor^® 488 goat anti-mouse IgG (H+L) used at a 1/1000 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). This antibody also gave a positive IF result in Hek293, HepG2 and MCF7 cells.

HL-60 (human promyelocytic leukemia cell line) cells were stained with 1 µg/mL ab14715 (blue) or an equal amount of an isotype control antibody (red) and analyzed by flow cytometry.