All lanes : HRP Anti-SDHA antibody [2E3GC12FB2AE2] (ab198493) at 1/5000 dilution

Lane 1 : Wild-type HEK-293 (human epithelial cell line from embryonic kidney) whole cell lysate
Lane 2 : SDHA knockout HEK-293 whole cell lysate
Lane 3 : MCF7 (human breast adenocarcinoma cell line) whole cell lysate
Lane 4 : HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 70 kDa

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.

All lanes : Anti-SDHA antibody [2E3GC12FB2AE2] (ab14715)

Lane 1 : Isolated mitochondria from Human heart at 5 µg
Lane 2 : Isolated mitochondria from Bovine heart at 4 µg
Lane 3 : Isolated mitochondria from Rat heart at 10 µg
Lane 4 : Isolated mitochondria from Mouse heart at 10 µg
Lane 5 : Isolated mitochondria from HepG2 (human liver hepatocellular carcinoma cell line) at 20 µg

Predicted band size: 70 kDa
Observed band size: 70 kDa

ab14715 (2µg/ml) staining SDHA in human testis using an automated system (DAKO Autostainer Plus). Using this protocol there is cytoplasmic and mitochondrial staining within the seminal vesicles.
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

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.