Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2]

12 product images

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  Li et al PLoS One. 2016 Feb 22;11(2):e0149728. doi: 10.1371/journal.pone.0149728. eCollection 2016. Fig 5.

  Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] (ab14734)

  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

  All lanes: Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] (ab14734)

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  Flow Cytometry - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] (ab14734)

  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 22°C. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (Mouse IgG2b [PLPV219] - Isotype Control 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.
  
  

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  Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] (ab14734)

  Lanes 1-4: Merged signal (red and green). Green - ab14734 observed at 17 kDa. Red - loading control, Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 observed at 37 kDa.
  

  ab14734 was shown to react with VDAC1 / Porin in HEK-293T wildtype. Loss of signal was observed when knockout sample Human VDAC1 (Porin) knockout HEK-293T 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 (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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (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.

  All lanes: Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] (ab14734) at 1/1000 dilution

  Lane 1: Wild-type Hap1 cell lysate at 20 µg

  Lane 2: VDAC1 knockout Hap1 cell lysate at 20 µg

  Lane 3: Wild-type HEK-293T cell lysate at 20 µg

  Lane 4: VDAC1 knockout HEK-293T cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772) at 1/20000 dilution

  Predicted band size: 31 kDa

  Observed band size: 17 kDa, 37 kDa

  Lanes 1 - 4: Merged signal (red and green). Green - ab14734 observed at 31 kDa. Red - loading control, Anti-GAPDH antibody [EPR16891] - 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 Human VDAC1 (Porin) knockout HEK-293T cell line ab255444 (knockout cell lysate Human VDAC1 (Porin) knockout HEK-293T 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 (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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (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.

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  This image is courtesy of a customer review by Michiel Krols.

  Immunocytochemistry/ Immunofluorescence - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] (ab14734)

  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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  Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] (ab14734)

  Diluting buffer: 5% NFDM /TBST
  

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

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

  All lanes: Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] (ab14734) at 1/1000 dilution

  Lane 1: N-GST tagged Recombinant Human VDAC1 (aa1 to 283) protein at 0.01 µg

  Lane 2: N-GST tagged Recombinant Human VDAC2 (aa 1 to 294) protein at 0.01 µg

  Lane 3: N-GST tagged Recombinant Human VDAC3 (aa 1 to 283) protein at 0.01 µg

  Observed band size: 33 kDa

  Exposure time: 10s

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  Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] (ab14734)

  Blocking and dilution buffer: 5% NFDM /TBST.

  All lanes: Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] (ab14734) at 1/1000 dilution

  Lane 1: Wild type HAP1 whole cell lysate at 20 µg

  Lane 2: VDAC1 knockout HAP1 whole cell lysate at 20 µg

  Observed band size: 33 kDa

  Exposure time: 5s

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  Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] (ab14734)

  Diluting buffer: 5% NFDM /TBST

  All lanes: Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] (ab14734) at 1/1000 dilution

  Lane 1: HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate at 20 µg

  Lane 2: HAP1 (Wildtype control Human chronic myelogenous leukemia near-haploid cell line) whole cell lysate at 20 µg

  Lane 3: MEF (Mus musculus Embryo Fibroblast) whole cell lysate at 20 µg

  Lane 4: Rat brain tissue lysate at 20 µg

  Observed band size: 33 kDa

  Exposure time: 5s

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  Sun Y et al Voltage-dependent anion channels (VDACs) recruit Parkin to defective mitochondria to promote mitochondrial autophagy. J Biol Chem. 2012 Nov 23;287(48):40652-60. doi: 10.1074/jbc.M112.419721.

  Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] (ab14734)

  (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.

  All lanes: Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] (ab14734)

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  Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] (ab14734)

  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.

  All lanes: Western blot - Anti-VDAC1/Porin + VDAC3 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

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  Immunocytochemistry/ Immunofluorescence - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] (ab14734)

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

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  Image courtesy of an anonymous customer review.

  Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] (ab14734)

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

  All lanes: Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] (ab14734) at 1/500 dilution

  All lanes: MCF7 (Human breast adenocarcinoma cell line) cell lysate at 10 µg

  Secondary

  All lanes: HRP-conjugated goat anti-mouse polyclonal at 1/5000 dilution

  Developed using the ECL technique.

  Performed under reducing conditions.

  Exposure time: 3min

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  This image is courtesy of an anonymous customer review

  Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] (ab14734)

  All lanes: Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] (ab14734) at 1/5000 dilution

  Lanes 1 - 2: Rat brain cell lysate (homogenate) at 30 µg

  Lanes 3 - 4: Rat brain cell lysate (mitochondrial) at 30 µg

  Secondary

  All lanes: HRP conjugated sheep anti-mouse IgG

  Developed using the ECL technique.

  Performed under reducing conditions.

  Observed band size: 39 kDa

  Exposure time: 5min