Immunocytochemistry/ Immunofluorescence - VDAC1 / Porin antibody [20B12] - Mitochondrial Loading Control (ab14734)

Immunofluoresence using ab14734 at 0.2 µg/ml on human fibroblasts (red).
Nuclei were labelled with DAPI (blue).

Western blot - VDAC1 / Porin antibody [20B12] - Mitochondrial Loading Control (ab14734)

All lanes : Anti-VDAC1 / Porin antibody [20B12AF2] - Mitochondrial Loading Control (ab14734) at 1/5000 dilution

Lane 1 : Rat brain cell lysate (homogenate)
Lane 2 : Rat brain cell lysate (homogenate)
Lane 3 : Rat brain cell lysate (mitochondrial)
Lane 4 : Rat brain cell lysate (mitochondrial)

Lysates/proteins at 30 µg per lane.

Secondary
HRP conjugated sheep anti-mouse IgG
developed using the ECL technique

Performed under reducing conditions.

Observed band size : 39 kDa (why is the actual band size different from the predicted?)


Exposure time : 5 minutes

This image is courtesy of an anonymous Abreview

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - VDAC1 / Porin antibody [20B12] - Mitochondrial Loading Control (ab14734)

Ab14734 staining Human normal left ventricle. Staining is localized to the cytoplasm.
Left panel: with primary antibody at 2 ug/ml. Right panel: isotype control.
Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 AR buffer citrate pH 6.0 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 Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

Flow Cytometry - VDAC1 / Porin antibody [20B12] - Mitochondrial Loading Control (ab14734)

Overlay histogram showing HepG2 cells stained with ab14734 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab14734, 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 IgG2b [PLPV219] (ab91366, 2µg/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 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

Western blot - VDAC1 / Porin antibody [20B12] - Mitochondrial Loading Control (ab14734)

All lanes : Anti-VDAC1 / Porin antibody [20B12AF2] - Mitochondrial Loading Control (ab14734)

Lane 1 : Isolated mitochondria from human heart at 15 µg
Lane 2 : Isolated mitochondria from bovine heart at 6 µg
Lane 3 : Isolated mitochondria from rat heart at 30 µg
Lane 4 : Isolated mitochondria from mouse heart at 30 µg
Lane 5 : HepG2 at 30 µg


Observed band size : 37 kDa (why is the actual band size different from the predicted?)


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