Anti-SDHA 抗体 [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.

Mitochondrial and cytoplasmic fractions were obtained from whole flies with Abcam Mitochondrial Isolation Kit for Tissue (ab110168) and from robin fibroblast and mouse hepatocyte cell lines with Abcam Mitochondrial Isolation Kit for Cells (ab110170).

ab14715 was used to stain SDHA at 0.1 μg/ml

HRP-conjugated Horse Anti-mouse IgG at 1 μg/ml was used as a secondary antibody. 

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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.