All lanes : Anti-Cytochrome C antibody (ab90529) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
Lane 3 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate
Lane 4 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
Lane 5 : Human testis tissue lysate - total protein (ab30257)
Lane 6 : Human liver tissue lysate - total protein (ab29889)

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 12 kDa
Observed band size: 15 kDa why is the actual band size different from the predicted?


Exposure time: 30 minutes

ab90529 staining cytochrome C in HepG2 cells treated with mevastatin sodium salt (ab120854), by ICC/IF. Increase of cytochrome C expression correlates with increased concentration of mevastatin sodium salt, as described in literature.
The cells were incubated at 37°C for 4 hours in media containing different concentrations of ab120854 (mevastatin sodium salt) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab90529 (1 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

ab90529 staining cytochrome C in HepG2 cells treated with atorvastatin hemicalcium salt (ab141083), by ICC/IF. Increase of cytochrome C expression correlates with increased concentration of atorvastatin hemicalcium salt, as described in literature.
The cells were incubated at 37°C for 4 hours in media containing different concentrations of ab141083 (atorvastatin hemicalcium salt) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab90529 (1 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

ab90529 staining cytochrome C in HepG2 cells treated with mevastatin (ab120652), by ICC/IF. Increase of cytochrome C expression correlates with increased concentration of mevastatin, as described in literature.
The cells were incubated at 37°C for 4 hours in media containing different concentrations of ab120652 (mevastatin) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab90529 (1 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

ab90529 staining cytochrome C in HepG2 cells treated with fluvastatin sodium salt (ab120651), by ICC/IF. Increase of cytochrome C expression correlates with increased concentration of fluvastatin sodium salt, as described in literature.
The cells were incubated at 37°C for 48 hours in media containing different concentrations of ab120651 (fluvastatin sodium salt) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab90529 (1 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

ICC/IF image of ab90529 stained HepG2 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 ab90529 at 1µg/ml overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti- rabbit 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. This antibody also gave a positive result in HeLa, Hek293, and MCF-7 formaldehyde (4%) (10min) fixed cells.

All lanes : Anti-Cytochrome C antibody (ab90529) at 1 µg/ml

Lane 1 : Heart (Mouse) Tissue Lysate
Lane 2 : Heart (Rat) Tissue Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 12 kDa
Observed band size: 14 kDa why is the actual band size different from the predicted?
Additional bands at: 30 kDa, 65 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 4 minutes

IHC image of Cytochrome C staining in human skeletal muscle formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab90529, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.