Anti-VDAC1/Porin + VDAC3抗体[20B12AF2] (ab14734)

Lanes 1 - 4: Merged signal (red and green). Green - ab14734 observed at 31 kDa. Red - loading control, ab181602 observed at 37 kDa. The lower band very close to VDAC1 is likely to be VDAC3.

ab14734 was shown to react with VDAC1/Porin + VDAC3 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-Mouse IgG H&L (IRDye^® 800CW) preadsorbed and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

ab14734 staining VDAC1/Porin + VDAC3 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.

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Blocking and diluting buffer and concentration: 5% NFDM/TBST.

N-GST tagged Recombinant Human VDAC1 protein is available as ab132481

N-GST tagged Recombinant Human VDAC2 protein is available as ab152793

Blocking and dilution buffer: 5% NFDM /TBST.

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

(A) The Anti-VDAC1/Porin + VDAC3 antibody recognizes VDAC1 and VDAC3 but not VDAC2.

Lysates from wild-type MEFs and VDAC1/3-/- MEFs were analyzed by western blotting with Anti-VDAC1/Porin + VDAC3 (top panel) or anti Tom20 antibodies (bottom panel).

(B) The Anti-VDAC1/Porin + VDAC3 antibody does not recognize VDAC2 in VDAC1/3-/- MEFs.

HA-tagged VDAC1, 2 or 3 were expressed in VDAC1/3-/- MEFs, immunoprecipitated with anti-HA antibodies and analyzed by western blotting with anti-HA antibodies (top panel) or Anti-VDAC1/Porin + VDAC3 antibodies (bottom panel). The Anti-VDAC1/Porin + VDAC3 antibodies recognize HA-VDAC1 and HA-VDAC3, but not HA-VDAC2 . Note that HA-VDAC3 migrates at a lower molecular weight than HA-VDAC1 and HA-VDAC2 .

Molecular weights are indicated in kDa.

Immunoblot analysis of endogenous VDAC1/Porin and VDAC3 in subcellular fractions of MA-10 cell lysates. VDAC1/Porin and VDAC3 are used as a mitochondrial marker proteins.

Lane 1: Whole cell lysate

Lane 2: Cytosolic fraction

Lane 3: Mitochondrial-enriched fraction

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).

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

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