All lanes : Anti-COX IV antibody [mAbcam33985] - Mitochondrial Marker (ab33985) at 1 µg/ml

Lane 1 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
Lane 2 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
Lane 3 : ab29330
Lane 4 : Skeletal Muscle (Mouse) Tissue Lysate
Lane 5 : ab29073

Lysates/proteins at 10 µg per lane.

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

Performed under reducing conditions.

Predicted band size: 19 kDa
Observed band size: 15 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-arginine, and sodium azide (ab33985).

ICC/IF image of ab33985 stained human HeLa cells. The cells were PFA fixed (10 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab33985, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. 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). DAPI was used to stain the cell nuclei (blue).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-arginine, and sodium azide (ab33985).

Ab33985 staining COX IV in HeLa (Human cervix adenocarcinoma epithelial cell) cells by ICC/IF (Immunocytochemistry/Immunofluorescence). Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% TritonX-100. Samples were incubated with primary antibody at 1:1000 dilution (1µg/ml). An Alexa Fluor^®594 Goat anti-mouse (ab150120) was used as a secondary antibody at 1:1000 dilution (2 µg/ml). Cells were counterstained with anti-Cyclophilin F (ab231155, 5.5µg/ml) and AlexaFluor^®488 Goat anti-Rabbit (ab150077, 2µg/ml).  DAPI was used as a nuclear counterstain. Ab33985 was used for negative control 1 at 1:1000 dilution (1µg/ml). For negative control 2, ab231155 was used at a 1:100 dilution (5.5µg/ml) and ab150129 was used as a secondary antibody at 1:1000 dilution (2µg/ml). Confocal image showing mitochondrial staining in HeLa cell line. 

This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-arginine, and sodium azide (ab33985).

Overlay histogram showing HeLa cells stained with ab33985 (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 (ab33985, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-arginine, and sodium azide (ab33985).