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

• WB

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Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] - Mitochondrial Loading Control (AB14734)

Lanes 1-4 : Merged signal (red and green). Green - ab14734 observed at 17 kDa. Red - 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 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.

All lanes:

Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] - Mitochondrial Loading Control (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 2:

Western blot - Human VDAC1 (Porin) knockout HEK-293T cell line (<a href='/products/cell-lines/human-vdac1-porin-knockout-hek-293t-cell-line-ab255444'>ab255444</a>)

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 (<a href='/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-800cw-preadsorbed-ab216772'>ab216772</a>) at 1/20000 dilution

Predicted band size: 31 kDa

Observed band size: 17 kDa,37 kDa

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• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] - Mitochondrial Loading Control (AB14734)

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

• ICC/IF

AbReview53174****

Immunocytochemistry/ Immunofluorescence - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] - Mitochondrial Loading Control (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.

This image is courtesy of an Abreview by Michiel Krols.

• Flow Cyt

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Flow Cytometry - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] - Mitochondrial Loading Control (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) (ab96879) at 1/500 dilution for 30 min at 22°C. 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.

• WB

Lab

Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] - Mitochondrial Loading Control (AB14734)

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

All lanes:

Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] - Mitochondrial Loading Control (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

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Exposure time: 5s

• WB

Lab

Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] - Mitochondrial Loading Control (AB14734)

Blocking and dilution buffer : 5% NFDM /TBST.

All lanes:

Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] - Mitochondrial Loading Control (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

false

Exposure time: 5s

• WB

Lab

Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] - Mitochondrial Loading Control (AB14734)

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

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

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

All lanes:

Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] - Mitochondrial Loading Control (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

false

Exposure time: 10s

• WB

AbReview52127****

Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] - Mitochondrial Loading Control (AB14734)

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

All lanes:

Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] - Mitochondrial Loading Control (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

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Exposure time: 3min

Image courtesy of an anonymous abreview.

• WB

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Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] - Mitochondrial Loading Control (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] - 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 (Human liver hepatocellular carcinoma cell line) cell lysate at 30 µg

Observed band size: 37 kDa

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• WB

PubMed

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

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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.

• WB

PubMed

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

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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.

• WB

AbReview7728****

Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] - Mitochondrial Loading Control (AB14734)

All lanes:

Western blot - Anti-VDAC1/Porin + VDAC3 antibody [20B12AF2] - Mitochondrial Loading Control (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

Observed band size: 39 kDa

true

Exposure time: 5min

This image is courtesy of an anonymous Abreview