ab14714 staining SDHB in wild-type HEK293 cells (top panel) and SDHB knockout HEK293 cells (bottom panel). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab14714 at 5µg concentration and ab6046 (Rabbit polyclonal to beta Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to mouse IgG (Alexa Fluor® 488) (ab150117) at 2 μg/ml (shown in green) and a goat secondary antibody to rabbit IgG (Alexa Fluor® 594) (ab150080) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

All lanes : Anti-SDHB antibody [21A11AE7] (ab14714) at 5 µg/ml

Lane 1 : Isolated mitochondria from Human heart at 5 µg
Lane 2 : Isolated mitochondria from Bovine Heart at 1 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) cells at 20 µg

Secondary
All lanes : Goat anti-Mouse secondary

Observed band size: 28 kDa why is the actual band size different from the predicted?
Additional bands at: 55 kDa. We are unsure as to the identity of these extra bands.



Extra bands in the mouse sample (lane 4) are due to the reaction of the IgG-specific goat anti-mouse secondary antibody with residual mouse blood in the heart tissue, as it is very difficult to entirely remove the blood from these small organs.

ab14714 staining SDHB in normal ageing human colon tissue by Immunohistochemistry (Frozen sections).

Mitochondrial localization of complex II visualized by immunocytochemistry using anti-complex II subunit 30 kDa Ip mAb 21A11 (ab14714). Cells were fixed, permeabilized and then labeled with ab14714 followed by an Alexa Fluor® 488-conjugated-goat-anti-mouse IgG2a isotype specific secondary antibody.

Overlay histogram showing HEK-293 (human epithelial cell line from embryonic kidney) cells stained with ab14714 (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 followed by the antibody (ab14714, 1µg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

ICC/IF image of ab14714 stained HeLa (human epithelial cell line from cervix adenocarcinoma) cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab14714, 1µg/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) at a concentration of 1.43µM.