Anti-SDHA antibody [2E3GC12FB2AE2]

• WB

Lab

Western blot - 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.

All lanes:

Western blot - Anti-SDHA antibody [2E3GC12FB2AE2] (ab14715) at 1/5000 dilution

Lane 1:

Wild-type HEK-293 (human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 2:

SDHA knockout HEK-293 whole cell lysate at 20 µg

Lane 3:

MCF7 (human breast adenocarcinoma cell line) whole cell lysate at 20 µg

Lane 4:

HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate at 20 µg

Predicted band size: 72 kDa

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• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SDHA antibody [2E3GC12FB2AE2] (AB14715)

Immunohistochemical analysis of formalin fixed paraffin embedded human testis labelling SDHA with ab14715 at a concentration of 0.1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab14715 Anti-SDHA antibody [2E3GC12FB2AE2] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-SDHA antibody [2E3GC12FB2AE2] (AB14715)

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

Lab

Immunocytochemistry - Anti-SDHA antibody [2E3GC12FB2AE2] (AB14715)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SDHA antibody [2E3GC12FB2AE2] (AB14715)

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.

• Flow Cyt

Unknown

Flow Cytometry - Anti-SDHA antibody [2E3GC12FB2AE2] (AB14715)

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.

• ICC

Supplier Data

Immunocytochemistry - Anti-SDHA antibody [2E3GC12FB2AE2] (AB14715)

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).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SDHA antibody [2E3GC12FB2AE2] (AB14715)

Immunohistochemical analysis of formalin fixed paraffin embedded human heart labelling SDHA with ab14715 at a concentration of 0.1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab14715 Anti-SDHA antibody [2E3GC12FB2AE2] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• WB

Unknown

Western blot - Anti-SDHA antibody [2E3GC12FB2AE2] (AB14715)

All lanes:

Western blot - 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: 72 kDa

Observed band size: 70 kDa

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