Flow Cytometry - COX IV antibody [20E8] - Mitochondrial Loading Control (ab14744)

Overlay histogram showing HeLa cells stained with ab14744 (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 (ab14744, 0.5µg/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.

Western blot - COX IV antibody [20E8] - Mitochondrial Loading Control (ab14744)

All lanes : Anti-COX IV antibody [20E8C12] - Mitochondrial Loading Control (ab14744) at 1/5000 dilution

Lane 1 : Cytoplasmic fraction mouse NIH/3T3 cell lysate
Lane 2 : Nuclear fraction mouse NIH/3T3 cell lysate

Secondary
HRP conjugated goat anti-mouse antibody
developed using the ECL technique

Performed under reducing conditions.

Observed band size : 16 kDa (why is the actual band size different from the predicted?)


Exposure time : 3 minutes

This image is courtesy of an Abreview submitted by Camilla Skjerpen on 4 July 2005.

Immunocytochemistry/ Immunofluorescence - COX IV antibody [20E8] - Mitochondrial Loading Control (ab14744)

ab14744 at 1/100 staining MCF-10A cells (human mammary epithelial cell line) by ICC/IF. The cells were methanol/acetone fixed at -20C for 5 minutes, blocked with BSA and then incubated with the antibody for 16 hours. The positive tissue was colocalised with a mitotracker (mitotrackers are a series of patented mt-selective stains that are concentrated by active mt and well retained during cell fixation). An Alexa-Fluor ® 488 conjugated goat anti-mouse antibody was used as the secondary. The image shows COXIV staining in green and DAPI staining in blue.

This image is courtesy of an anonymous Abreview

Immunohistochemistry (PFA perfusion fixed frozen sections) - COX IV antibody [20E8] - Mitochondrial Loading Control (ab14744)

ab14744 staining COX IV in rat brain tissue sections by Immunohistochemistry (PFA perfusion fixed frozen sections). Rats were anesthetized and intracardially perfused with 500 ml of normal saline at room temperature, followed by 500 ml of ice-cold, freshly made 4% paraformaldehyde in phosphate buffer (PB, 0.1 M, pH 7.4). A Cy3 conjugated anti mouse antibody was used as secondary. 

Image from Hashimoto T. et. al., PLoS ONE. 2008; 3(8): e2915 (Fig. 1B)

Western blot - COX IV antibody [20E8] - Mitochondrial Loading Control (ab14744)

All lanes : Anti-COX IV antibody [20E8C12] - Mitochondrial Loading Control (ab14744) at 1 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 murine heart at 10 µg


Observed band size : 16 kDa (why is the actual band size different from the predicted?)