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

Lane 1 : Wild-type Hap1 cell lysate
Lane 2 : VDAC1 knockout Hap1 cell lysate
Lane 3 : Wild-type HEK-293T cell lysate
Lane 4 : VDAC1 knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Lanes 1 - 4: Merged signal (red and green). Green - ab14734 observed at 31 kDa. Red - loading control, ab181602 observed at 37 kDa.  

 ab14734 was shown to react with VDAC1 / Porin in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab255444 (knockout cell lysate ab263839) was used. Wild-type and VDAC1 / Porin knockout samples were subjected to SDS-PAGE. ab14734 and Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

 

Double immunofluorescent staining and immunoblot analysis of the endogenous SCPX and SCP2 proteins in MA-10 cells.

Immunoblot analysis of endogenous SCP2 in subcellular fractions of MA-10 cell lysates. VDAC1 is used as a mitochondrial marker protein, and GAPDH is used as a whole cell lysate loading control.

Lane 1: Whole cell lysate

Lane 2: Cytosolic fraction

Lane 3: Mitochondrial-enriched fraction

All lanes : Anti-VDAC1 / Porin antibody [20B12AF2] (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 (Human liver hepatocellular carcinoma cell line) cell lysate 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.

ab14734 staining VDAC1 / Porin in human HeLa (Human epithelial cell line from cervix adenocarcinoma) cells by ICC/IF (Immunocytochemistry/immunofluorescence).

Cells were fixed with paraformaldehyde, permeabilized with 0.1% Triton X-100 for 3 minutes and blocked with 0.2% serum for 60 minutes at 22°C. Samples were incubated with primary antibody (1/200 in 0.5% BSA and 0.02% Triton X100 in PBS) for 16 hours at 4°C. An FITC-conjugated goat anti-mouse IgG polyclonal was used as the secondary antibody at a dilution of 1/200.

See Abreview

Overlay histogram showing HepG2 (Human liver hepatocellular carcinoma cell line) 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, 1 µg/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, 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 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

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

Anti-VDAC1 / Porin antibody [20B12AF2] (ab14734) at 1/500 dilution + MCF7 (Human breast adenocarcinoma cell line) cell lysate at 10 µg

Secondary
HRP-conjugated goat anti-mouse polyclonal at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Exposure time: 3 minutes

0.5% TBS-tween + Lait 5% NaN3 for 16 hours at 4ºC.

See Abreview

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

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

Lysates/proteins at 30 µg per lane.

Secondary
All lanes : 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

See Abreview