Testes of bb8^ms mutants show defects in post-meiotic, elongated spermatids.

(A-B) Spermatids from WT (A) and bb8^ms (B) testis both have elongated cysts, but there are large spherical vesicles in the mutant (arrows) by phase contrast microscopy. Scale bars: 100 μm.

(C, D) Mitochondria of elongated spermatids stained with ATP5α antibody ab14748 in WT (C) and in bb8^ms (D) mutants. ATP5α positive staining of the large vesicles in the cysts are indicated by arrow. Scale bars: 50 μm.

(E, F) JC-1 staining positive large vesicles (arrows) are absent from WT (E), but present in bb8^ms elongated cysts (F). Scale bars: 25 μm.

Western blots were probed for HNE-damaged mitochondrial protein from brainstem (A), cerebellum (B) and “rest” brain (R). The sample designation indicates the age group (y for P25-P35, o for P45-P55), the genotype (KO, Ctrl for controls) and a number to distinguish independent samples.

Panel D is the blot from panel A reprobed for the mitochondrial marker ATPase (ATP5a) using ab14748 to demonstrate that extended sample storage did not degrade sample protein in general. Black lines in the MW lanes are magic marker on the film to indicate the positions of the prestained molecular weight standards on the blot.

For full image please see paper.

All lanes : Anti-ATP5A antibody [15H4C4] - Mitochondrial Marker (ab14748) at 1 µg/ml

Lane 1 : Isolated mitochondria from human heart at 10 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 : HepG2 (Human liver hepatocellular carcinoma cell line) lysate at 20 µg

All lanes : Anti-ATP5A antibody [15H4C4] - Mitochondrial Marker (ab14748) at 1 µg/ml

Lane 1 : Human liver tissue lysate
Lane 2 : HepG2 (Human hepatocellular liver carcinoma cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Observed band size: 55 kDa why is the actual band size different from the predicted?
Additional bands at: 36 kDa. We are unsure as to the identity of these extra bands.

ICC/IF image of ab14748 stained HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

The cells were fixed in 100% methanol (5 minutes), 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 1 hour. The cells were then incubated overnight at +4°C with ab14748 at 1 µg/ml (shown in red) and ab6160 (Rat monoclonal to Tubulin) at 1 µg/ml (shown in green).

This was followed by an incubation at room temperature for 1 hour with ab150119, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 647) preadsorbed, at 0.5 µg/ml (shown in red) and ab150165, Goat Anti-Rat IgG H&L (Alexa Fluor^® 488) preadsorbed, at 0.5 µg/ml (shown in green).

Nuclear DNA was labeled with DAPI (shown in blue).

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

This product also gave a positive signal under the same testing conditions in HeLa cells fixed with 4% formaldehyde (10 minutes).

ab14748 staining ATP5A in MDA-MB-231 cells by ICC/IF (Immunocytochemistry/immunofluorescence).

Cells were fixed with formaldehyde, permeabilized with 1% Triton X-100 and blocked with 10% BSA for 1 hour at 21°C. Samples were incubated with primary antibody (1/100 in BSA + 0.02% Tween 20) for 1 hour at 21°C. A DyLight^® 550-conjugated goat anti-mouse IgG polyclonal  (1/500) was used as the secondary antibody.

See Abreview

ICC/IF image of ab14748 stained MCF7 (Human breast adenocarcinoma cell line) cells.

The cells were fixed in 4% formaldehyde (10 minutes) and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab14748, 10 µg/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 1 hour. Alexa Fluor^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1 hour. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43 µM.

ab14748 (1 µg/ml) staining ATP5A in human heart (left ventricle), using an automated system (DAKO Autostainer Plus). Using this protocol there is mitochondrial staining of cardiomyocytes.
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH 6.1 in a DAKO PT link. Slides were peroxidase blocked in 3% H₂O₂ in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes.

Slides were counterstained with hematoxylin 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.

Overlay histogram showing HepG2 (Human liver hepatocellular carcinoma cell line) cells stained with ab14748 (red line).

The cells were fixed with 4% paraformaldehyde (10 minutes) and then permeabilized with 0.1% PBS-Tween for 20 minutes. 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 (ab14748, 1 µg/1x10⁶ cells) for 30 minutes at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 minutes at 22ºC. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2 µg/1x10⁶ cells) used under the same conditions.

Acquisition of >5,000 events was performed.

This antibody gave a positive signal in HepG2 cells fixed with 80% methanol (5 minutes)/permeabilized in 0.1% PBS-Tween used under the same conditions.