Vdac1 porin

Manufactured by Abcam

Sourced in United States

VDAC1/Porin is a protein that forms a voltage-dependent anion channel in the outer mitochondrial membrane. It is involved in the regulation of metabolite transport across the mitochondrial membrane.

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Lab products found in correlation

β actin, by Merck Group (4 mentions) Nupage, by Thermo Fisher Scientific (3 mentions) Cyt c, by Abcam (2 mentions) Dc kit, by Bio-Rad (2 mentions) Complete protease inhibitor mixture, by Roche (2 mentions) Chemidoc system, by Bio-Rad (2 mentions) Digitonin, by Roche (1 mentions) Anti bax antibody, by Cell Signaling Technology (1 mentions) Complete protease inhibitor, by Roche (1 mentions) Anti cytochrome c antibody, by BD (1 mentions) Odyssey blocking buffer tbs, by LI COR (1 mentions) Novex bis tris gel, by Thermo Fisher Scientific (1 mentions) Odyssey nitrocellulose membrane, by LI COR (1 mentions) Odyssey fc imaging system, by LI COR (1 mentions) Phospho stat3 tyr705, by Cell Signaling Technology (1 mentions) Phospho stat3 ser727, by Cell Signaling Technology (1 mentions) Stat3, by Cell Signaling Technology (1 mentions) Vinculin, by Bio-Rad (1 mentions) γ tubulin, by Bio-Rad (1 mentions) Ndufb8, by Abcam (1 mentions)

Spelling variants of this product

VDAC1 (57 citations) Porin (15 citations) Anti-VDAC1/Porin (8 citations) Rabbit anti-VDAC1/Porin (2 citations)

10 protocols using vdac1 porin

Quantification of BAX protein

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Cells (1 × 10⁶ cells/well) were seeded in 6-well clear bottom plate and incubated with serial dilutions of BTSA1 or vehicle (0.2% DMSO) in media with no FBS in a final volume of 2000 μL. After 2 hr, FBS was supplemented to a final concentration of 10%. Following 4 hr treatment, cells were lysed in 100 μL of Digitonin buffer [20 mM Hepes, pH 7.2, 10 mM KCl, 5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 250 mM sucrose, 0.025% Digitonin (from 5% w/v stock) and complete protease inhibitors (Roche Applied Science)] and incubated on ice for 10 min. The supernatants were isolated by centrifugation at 15,000 x g for 10 min and the mitochondrial pellets solubilized in 1% Triton X-100/PBS for 1 hr at 4 °C. Pellets were solubilized, subjected to a 15,000 x rpm spin for 10 min, and 50 ng of protein was mixed with 25 μl LDS/DTT loading buffer. The equivalent fractional volume of the corresponding supernatant samples was mixed with 25 μl LDS/DTT loading buffer. The mitochondrial supernatant and pellet fractions were then separated by 4–12% NuPage (Life Technologies) gels, and analyzed by immunoblotting with anti-BAX antibody (2772S, Cell Signaling). VDAC1/Porin (Abcam Cat. 15895) and β-Actin (Sigma Cat. A1978) are used for loading control of mitochondrial and supernatant fractions respectively.

Reyna D.E., Garner T.P., Lopez A., Kopp F., Choudhary G.S., Sridharan A., Narayanagari S.R., Mitchell K., Dong B., Bartholdy B.A., Walensky L.D., Verma A., Steidl U, & Gavathiotis E. (2017). Direct activation of BAX by BTSA1 overcomes apoptosis resistance in acute myeloid leukemia. Cancer cell, 32(4), 490-505.e10.

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Mitochondrial Cytochrome c Release Assay

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Mitochondria from liver of WT mice (1mg/ml) were resuspended in experimental buffer (125 mM KCl, 10 mM Tris-MOPS [pH 7.4], 5 mM glutamate, 2.5 mM malate, 1 mM K3PO4, 0.1 mM EGTA-Tris [pH 7.4]) and treated with the indicated concentrations of BTSA1 and recombinant BAX protein (200 nM), singly and in combination, and incubated at room temperature for 90 min. The supernatants were isolated by centrifugation at 5500 x g for 10 min and the mitochondrial pellets solubilized in 1% Triton X-100/PBS. Mitochondrial supernatant and pellet fractions were separated by 4–12% NuPage (Life Technologies) gels and analyzed by immunoblotting with anti-cytochrome c antibody (BD Biosciences Cat. 556433). VDAC1/Porin (Abcam Cat. 15895) and β-Actin (Sigma Cat. A1978) are used for loading control of mitochondrial and supernatant fractions respectively.

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Western Blot Analysis of Duodenum

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Fresh duodenum from each mouse was placed in 65°C lysis buffer
(60 mM Tris pH ~6.8/10 mM EDTA/2% SDS) directly after euthanization.
Samples were heated at 65°C for 10 minutes and then spun down for 10
minutes at 3600 rpm at 4°C. Samples were stored at −80°C
until time of use. At time of western blot, DTT (final concentration of 0.12 mM)
was added to each sample (50 μg of total protein), followed by 10
minutes incubation at 65°C. Samples were resolved on a 4–12
% gradient Novex Bis-Tris gel (Invitrogen) and electrophoretically
transferred onto Odyssey nitrocellulose membranes (Li-Cor, Lincoln, NE).
Membranes were subsequently dried for 1 hour at room temperature, followed by
blocking with Odyssey TBS blocking buffer (Li-Cor) for 1 hour at room
temperature. Primary (rabbit anti-MYO5B, 1:500, Sigma Prestige cat#
HPA040902 and rabbit anti-VDAC1/porin, 1:1000, Abcam cat# ab15895) and
secondary antibodies (IRDye 800CW donkey anti-rabbit IgG, 1:15000, Li-Cor
cat# 925-32213) were diluted in the blocking buffer with 0.2%
Tween-20. Membranes were visualized using Odyssey Fc Imaging System
(Li-Cor).

Weis V.G., Knowles B.C., Choi E., Goldstein A.E., Williams J.A., Manning E.H., Roland J.T., Lapierre L.A, & Goldenring J.R. (2015). Loss of MYO5B in Mice Recapitulates Microvillus Inclusion Disease and Reveals an Apical Trafficking Pathway Distinct to Neonatal Duodenum. Cellular and Molecular Gastroenterology and Hepatology, 2(2), 131-157.

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Protein Detection in Cell Lysates

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Tissues lysis and both separation and revelation of proteins were performed as described previously [3 (link)]. The primary antibodies used for protein detection are: STAT3 (Cell Signaling, #4904), Phospho-STAT3 (Tyr705) (Cell signaling, #9145), Phospho-STAT3 (Ser727) (Cell signaling, #9134), FTO (Abcam, ab65366), Actin (Sigma, A5060), SET7/9 (Santa Cruz, sc-56774), VDAC1/Porin (Abcam, ab14734).

Bravard A., Vial G., Chauvin M.A., Rouillé Y., Bailleul B., Vidal H, & Rieusset J. (2014). FTO contributes to hepatic metabolism regulation through regulation of leptin action and STAT3 signalling in liver. Cell Communication and Signaling : CCS, 12, 4.

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Brain Region Protein Expression Analysis

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Mice were perfused with phosphate-buffered saline; brains were isolated and different regions were dissected and homogenized in PBS supplemented with complete protease inhibitor mixture (Roche) [13 (link)]. Protein concentrations were determined using the DC kit (Bio-Rad Laboratories). The homogenates were resolved on SDS-PAGE gels, transferred onto a polyvinylidene difluoride (PVDF) membrane, and hybridized with the antibodies raised against Cyt c (Abcam), Cox1 (Mitosciences), NDUFB8, SDHA, UQCRFS1/Rieske, VDAC1/Porin (Abcam); GFAP (Cell Signaling), TUJ1 (Chemicon), Bcl2, BAD, BAX (Cell Signaling); β-actin, γ-tubulin, vinculin (Sigma); SOD1, SOD2, GPX1 (Abgent).
Depending on the size of the related protein, bands were normalized for housekeeping gene (β-actin, γ-tubulin, vinculin) or for total protein loading (visualized by stain free technology, in the Chemidoc system, Biorad). As the same blots were used for hybridization of multiple antibodies, the following figures used the same loading control: hippocampus 4w (Bcl2 and TUJ1; SOD2 and GFAP), hippocampus 8w (Bcl2 and TUJ1; BAX and SOD2; BAD and GFAP), hippocampus 12w (Bcl2 and GFAP), cortex 4w (BAD, SOD2, and GFAP; Bcl2 and TUJ1), cortex 8w (BAX, SOD2, and GFAP; BAD and TUJ1; Bcl2 and GFAP), and cortex 12w (Bcl2 and GFAP).

Pinto M., Vempati U.D., Diaz F., Peralta S, & Moraes C.T. (2018). Ablation of Cytochrome c in Adult Forebrain Neurons Impairs Oxidative Phosphorylation Without Detectable Apoptosis. Molecular neurobiology, 56(5), 3722-3735.

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Brain Tissue Preparation and Western Blot

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Mice were perfused with phosphate-buffered saline, brains were isolated, and cortex was dissected and homogenized in PBS supplemented with cOmplete™ Protease Inhibitor Mixture (Roche) and Phosphatase inhibitor PhosSTOP™ (Roche) [12 (link)]. Protein concentrations were determined using the DC kit (Bio-Rad Laboratories). The homogenates were resolved on SDS-PAGE gels, transferred onto a polyvinylidene difluoride (PVDF) membrane, and hybridized with the antibodies raised against total OXPHOS rodent Cocktail, NDUFB8, NDUFA9, SDHA, UQCRFS1/RISP, VDAC1/Porin, CytC, COX1, ATP5a, UQCRC1/Core1, p62 (Abcam); GFAP, Synaptophysin, Nicastrin, Presenilin 1, LC3B, CHOP, Neurogranin (Cell Signaling); TUJ1 (Chemicon); Iba1 (Wako-016–20001); SOD2 (Upstate); APP C-Terminal Fragment, and Purified anti-β-Amyloid 1–16 Antibody clone 6E10 (Biolegend); 20s proteasome (Novus Biol). Bands were normalized for total protein loading (visualized by stain free technology, in the Chemidoc system, Biorad). As the same blots were used for hybridization of multiple antibodies, the following figures used the same loading control: Core1, VDAC1, Iba1 and p62; APP-fl, APP-Ct, and CHOP; Synaptophysin and Rieske; 20s proteasome and SOD2; 6E10 and LC3b.

Pinto M., Diaz F., Nissanka N., Guastucci C.S., Illiano P., Brambilla R, & Moraes C.T. (2022). Adult-onset deficiency of mitochondrial Complex III in a mouse model of Alzheimer’s disease decreases amyloid beta plaque formation. Molecular neurobiology, 59(10), 6552-6566.

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Mitochondrial Protein Isolation and Analysis

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Mitochondrial proteins were prepared from fresh isolated mitochondria solubilized in 20 mM HEPES, 2 mM EDTA, 0.5% Triton-X100, 150 mM NaCl, 1 mM PMSF, and a HALT protease cocktail inhibitor [30 (link)]. These were then buffered and fractionated in 8% Bis-Tris polyacrylamide gel with MOPS and a 5 mM sodium bisulfite running buffer before being transferred onto a 0.2 μm PVDF membrane with NUPAGE transfer buffer in the semidry apparatus. The antibodies PGC1-α+β (Cat # ab72230, Abcam), MFN2 (Cat # ab50843, Abcam), VDAC1/Porin (Cat # ab15895, Abcam), ceruloplasmin (Cat # ab8813, Abcam), and β-actin (Cat # ab8227, Abcam) were used according to a previous study [30 (link), 32 (link)]. Detection of carbonyl groups introduced into proteins by oxidative stress was performed with the OxyBlot Protein Oxidation Detection Kit (Cat # S7150 Millipore) according to [30 (link), 32 (link)]. The quantity of ceruloplasmin was evaluated in plasma samples, mixing 5 μL of plasma, 5 μL of sample buffer, and 15 μL of H₂O. The sample was boiled at 95°C for 10 min and centrifuged and run in a protein electrophoresis and blotted in PVDF membrane for ceruloplasmin immunodetection [30 (link)].

Ruiz L.M., Salazar C., Jensen E., Ruiz P.A., Tiznado W., Quintanilla R.A., Barreto M, & Elorza A.A. (2015). Quercetin Affects Erythropoiesis and Heart Mitochondrial Function in Mice. Oxidative Medicine and Cellular Longevity, 2015, 836301.

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Immunoblotting Analysis of Apoptosis-Related Proteins

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Protein lysates were obtained by cell lysis in Triton X-100 buffer (50 mM Tris-HCL pH 7.40, 150 mM NaCl, 5 mM MgCl₂, 1 mM EGTA, 10% Glycerol, 1% Triton X-100 [Sigma]). Protein samples were electrophoretically separated on 4–12% NuPage (Life Technologies) gels, transferred to mobilon-FL PVDF membranes (Millipore) and subjected to immunoblotting. For visualization of proteins with Odyssey Infrared Imaging System (LI-COR Biosciences) membranes were blocked in PBS containing 5% dry milk. Primary antibodies were incubated overnight at 4°C in a 1:1,000 dilution. After washing, membranes were incubated with an IRdye800-conjugated goat anti-rabbit IgG or IRdye800-conjugated goat anti-mouse IgG secondary antibodies (LI-COR Biosciences) in a 1:5,000 dilution. Proteins were detected with Odyssey Infrared Imaging System. Antibodies were used to detect the following proteins on membrane: BCL-X_L (BD Cat. 610747), MCL-1 (Cell Signaling Cat. 4572), BAX (Cell Signaling Cat. 2772), BCL-2 (Cell Signaling Cat. 4223), BAK (Millipore Cat. 06-536), Cytochrome c (BD Cat. 556433), VDAC1/Porin (Abcam Cat. 15895), β-Actin (Sigma Cat. A1978).

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Protein Expression Analysis in Cells

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RIPA lysis buffer was used to lysate cells.³⁴ Western blot analysis was performed using equal quantity of protein. Protein concentration was determined by Bradford method (Sigma‐Aldrich) and equal amounts were subjected to Western blot analysis. All membranes were incubated for 12 hours at 4°C with antibodies against sirt1, ERα, IGF1R, CCND1, vimentin, N‐cadherin, bax, bcl‐2, parp1 and cytochrome c (all from Santa Cruz Biotechnology, Santa Cruz CA, USA). Membranes were incubated with HRP‐conjugated secondary antibodies (Bethyl Laboratories, Montgomery, TX, USA), and immunoreactive bands were visualized with the ECL Western blotting detection system (Santa Cruz Biotechnology, Santa Cruz CA, USA). GAPDH (Santa Cruz Biotechnology, Santa Cruz CA, USA) or VDAC1/porin (Abcam, Cambridge, UK) antibodies were used as a loading control.

Chimento A., De Luca A., Nocito M.C., Sculco S., Avena P., La Padula D., Zavaglia L., Sirianni R., Casaburi I, & Pezzi V. (2021). SIRT1 is involved in adrenocortical cancer growth and motility. Journal of Cellular and Molecular Medicine, 25(8), 3856-3869.

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Western Blot Analysis of Mitochondrial Proteins

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Homogenates for western blot analyses were obtained from cell cultures. Cells were washed with PBS1x, homogenized with a 100 mL of cold lysis buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 2 mM sodium orthovanadate, 100 mM NaF, 20 mM sodium pyrophosphate, 1% NP-40, and protease inhibitors Mini protease tablet (Roche)], and centrifuged at 700 3 g for 10 min to remove nuclei and cellular debris. Proteins from total homogenates were resolved in 7.5%, 10% or 12.5% acrylamide gels for SDS/PAGE and transferred to Immobilon membranes (Millipore). The antibodies used included: PARP (Cell Signaling; 1/1,000 diluted), caspase 3 (Cell Signaling; 1/1,000 diluted), VDAC-1/Porin (Abcam; 1/5,000 diluted), MFN1 (Abcam; 1/1,000 diluted), MFN2 (Abcam, 1/1,000 diluted), OPA1 (BD Transduction Laboratories TM , 1/1,000 diluted), DRP1 (BD Transduction Laboratories TM , 1/1,000 diluted), phospho-DRP1 Ser616 (Cell Signaling, 1/1,000 diluted), FIS1 (BioVision Corporate Headquarters, 1/1,000 diluted), p70 S6 kinase (Cell Signaling; 1/1,000 diluted), Phospho-p70 S6 kinase (Cell Signaling; 1/1,000), LC3 (MBL, 1/1,000 diluted), p62 (Progen, 1/1,000 diluted), BNIP3 (Abcam, 1/1,000 diluted), b-actin (Sigma-Aldrich; 1/10,000 diluted), and a-tubulin (Sigma-Aldrich; 1/5,000 diluted). Proteins were detected by the ECL method and quantified by scanning densitometry.

Miret-Casals L., Sebastián D., Brea J., Rico-Leo E.M., Palacín M., Fernández-Salguero P.M., Loza M.I., Albericio F, & Zorzano A. (2018). Identification of New Activators of Mitochondrial Fusion Reveals a Link between Mitochondrial Morphology and Pyrimidine Metabolism. Cell chemical biology, 25(3).

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