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Total oxphos human wb antibody cocktail

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The Total OXPHOS Human WB Antibody Cocktail is a collection of antibodies designed to detect the five major protein complexes of the human oxidative phosphorylation (OXPHOS) system. This product is intended for use in western blotting applications.

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Lab products found in correlation

β actin, by Merck Group (3 mentions) Pvdf membrane, by Merck Group (3 mentions) Ab110411, by Abcam (2 mentions) β actin, by Cell Signaling Technology (2 mentions) Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Tom40, by Abcam (1 mentions) Hsp90, by Santa Cruz Biotechnology (1 mentions) Revert total protein stain, by LI COR (1 mentions) Anti 4e bp1, by Cell Signaling Technology (1 mentions) Anti hsp70, by Abcam (1 mentions) Anti hsp60, by Abcam (1 mentions) Anti ubiquitin, by Thermo Fisher Scientific (1 mentions) Anti smac, by Abcam (1 mentions) Anti vdac, by Santa Cruz Biotechnology (1 mentions) Anti phospho 4ebp1, by Cell Signaling Technology (1 mentions) Anti hsp90, by Enzo Life Sciences (1 mentions) Anti mia40, by Santa Cruz Biotechnology (1 mentions) Anti rps6, by Abcam (1 mentions) Anti phospho rps6, by Abcam (1 mentions) C myc, by Cell Signaling Technology (1 mentions)

Spelling variants of this product

OXPHOS cocktail (40 citations) Total OXPHOS (24 citations) OXPHOS antibody cocktail (23 citations) Total OXPHOS antibody cocktail (21 citations) Total OXPHOS cocktail (18 citations) MitoProfile Total OXPHOS Human WB Antibody cocktail (18 citations) Total OXPHOS human antibody cocktail (5 citations) Total OXPHOS WB antibody cocktail (4 citations) OXPHOS Human WB Antibody Cocktail (4 citations) Mouse anti-Total OXPHOS Human WB Antibody Cocktail (2 citations)

29 protocols using total oxphos human wb antibody cocktail

1

Western Blot Analysis of OXPHOS and DNA Damage

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Ten to 20 µg of protein per lane was separated by standard SDS–PAGE and transferred onto PVDF membranes. After blocking in blocking solution (BS; 100 mM Tris/HCl (pH 8.0), 450 mM NaCl, 5% dry milk, and 0.05% Tween-20), the membranes were incubated with antibodies against Total OXPHOS Human WB Antibody Cocktail (1:5000 in BS, Abcam), TOM40 (1:5000), p53 (1:5000 in BS, both from Santa Cruz Biotechnology, Dallas, TX, USA), and phospho-Ser139 Histone H2AX (γH2AX) (1:5000 in BS, Millipore/Merck KGaA, Darmstadt, Germany). Equal loading of whole cell lysates was verified by the detection of HSP90 (1:5000 in BS, Santa Cruz). Peroxidase-conjugated anti-mouse IgG (H + L) and anti-rabbit IgG (H + L) secondary antibodies (1:10,000; Abcam) were used and detection of specific signals was done with SuperSignal West Pico Chemiluminescent Substrate (Pierce/Thermo Scientific). Densitometric quantification was done with ImageJ (National Institutes of Health, Bethesda, MD, USA). Full immunoblot images are depicted in supplementary Fig. S4.
Marx C., Sonnemann J., Maddocks O.D., Marx-Blümel L., Beyer M., Hoelzer D., Thierbach R., Maletzki C., Linnebacher M., Heinzel T, & Krämer O.H. (2022). Global metabolic alterations in colorectal cancer cells during irinotecan-induced DNA replication stress. Cancer & Metabolism, 10, 10.
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2

Western Blot Antibodies for OXPHOS

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Antibodies used for Western blot included the Total OXPHOS Human WB Antibody Cocktail (#ab1104a1; Abcam), anti-Aco2 (#ab110321; Abcam), anti-4E-BP1 (#9644; Cell Signaling) and anti-phospho-4E-BP1 (#2855; Cell Signaling), anti-EF-1α1/2 (#sc-377439; Santa Cruz), anti-Hsp60 (#ab59457; Abcam), anti-Hsp70 (#ab47455; Abcam), anti-Hsp90 (#16F1; Enzo), anti-Mdh2 (#sc-293474; Santa Cruz), anti-Mia40 (#sc-365137; Santa Cruz), anti-rpS6 (#ab40820, Abcam); anti-phospho-rpS6 (#ab65748; Abcam), anti-SMAC (#ab8114; Abcam), anti-Tim22 (#14927-1-AP; Proteintech), anti-ubiquitin (#701339; Thermo Scientific), and anti-VDAC (#sc-390996; Santa Cruz). Total proteins were stained with REVERT Total Protein Stain (#926-11011; LI-COR).
Liu Y., Wang X., Coyne L.P., Yang Y., Qi Y., Middleton F.A, & Chen X.J. (2019). Mitochondrial carrier protein overloading and misfolding induce aggresomes and proteostatic adaptations in the cytosol. Molecular Biology of the Cell, 30(11), 1272-1284.
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3

Quantifying Protein Expression via Western Blot

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Specific protein expression in cell lines was determined by Western blot analysis as described before (Hlavac et al., 2019 (link); Pruss et al., 2020 (link)) using the following primary antibodies: Total OXPHOS human WB antibody cocktail (1:1,000, #ab110411, Abcam, Cambridge, U.K.), c-myc (1:1,000, #18583, clone E5Q6W; Cell Signaling Technology, Danvers, MA, United States), c-myc/n-myc (1:1,000, #13987, clone D3N8F; Cell Signaling Technology, Danvers, MA, United States) and β-actin (1:2,000, clone AC15; Sigma Aldrich, St. Louis, MO). Secondary HRP-linked antibodies were purchased from Cell Signaling Technology (#7076S, #7074S).
Dwucet A., Pruss M., Cao Q., Tanriover M., Prabhu V.V., Allen J.E., Peraud A., Westhoff M.A., Siegelin M.D., Wirtz C.R, & Karpel-Massler G. (2021). ONC201/TIC10 Is Empowered by 2-Deoxyglucose and Causes Metabolic Reprogramming in Medulloblastoma Cells in Vitro Independent of C-Myc Expression. Frontiers in Cell and Developmental Biology, 9, 734699.
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4

Mitochondrial Dynamics and Apoptosis Analysis

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Cell pellets were lysed in RIPA buffer containing 1× HaltTM protease inhibitor cocktail (78437, Thermo) and supernatants collected after 10 min centrifugation at 12000 × g for SDS-PAGE. Total proteins were transferred to PVDF membranes and blotted with the antibodies against FIS1 (ab71498, Abcam), MFN1 (ab104274, Abcam), β-Actin (ab8227, Abcam), Caspase-3 (9665, Cell Signaling), Caspase-8 (sc-5263, Santa Cruz), Caspase-9 (sc-8355, Santa Cruz), HSP70 (TA309356, Origene) and Total OXPHOS Human WB Antibody Cocktail (ab110411, Abcam).
Gonzalez-Ibanez A.M., Ruiz L.M., Jensen E., Echeverria C.A., Romero V., Stiles L., Shirihai O.S, & Elorza A.A. (2020). Erythroid Differentiation and Heme Biosynthesis Are Dependent on a Shift in the Balance of Mitochondrial Fusion and Fission Dynamics. Frontiers in Cell and Developmental Biology, 8, 592035.
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5

Molecular Mechanisms of EMT and Metabolism

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Recombinant human TNFα and TGFβ were purchased from R&D Systems (Minneapolis, MN, USA). Antibodies used are listed as follows: IkB, phospho-Smad2/3, total-Smad2/3, Snail, vimentin, lactate dehydrogenase-A, fatty acid synthase, stearoyl-CoA desaturase-1, phospho-acetyl-CoA carboxylase, total-acetyl-CoA carboxylase (Cell signaling, MA, USA); beta-actin, E-cadherin, N-cadherin (Santa Cruz, TX, USA); hexokinase II, pyruvate kinase isoform 2, fructose-bisphophotase-2, total OXPHOS Human WB Antibody Cocktail (Abcam, Cambridge, UK); Zeb1 (ProSci, CA, USA); and fructose-bisphophotase-1 (Abgent, Wuxi, China). All other reagents were from Sigma-Aldrich (MO, USA) unless stated otherwise.
Liu M., Quek L.E., Sultani G, & Turner N. (2016). Epithelial-mesenchymal transition induction is associated with augmented glucose uptake and lactate production in pancreatic ductal adenocarcinoma. Cancer & Metabolism, 4, 19.
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6

Protein Expression Analysis by Capillary Electrophoresis

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Specific protein expression in cell lines was determined by Western blot analysis or capillary electrophoresis as described before. Capillary electrophoresis was run on the Wes instrument (Proteinsimple, CA). The following antibodies were used on the Wes instrument: p-Akt (serine 473) (1:25, CST, Cell Signaling Technology, Danvers, MA), Akt (1:50, CST), Mcl-1 (1:50; CST:), Bcl-2 (1:25; R&D Systems), BIM (1:25; CST), Bcl-xL (1:25; CST), c-myc (1:25, CST), Usp9X (1:25; CST), Noxa (1:25, clone 114C307; Calbiochem), p-Akt (1:25, CST), Akt (1:25, CST), p-AMPK (1:25, CST), AMPK (1:25, CST), PHGDH antibody (Novus, #NBP1–87311), PSAT1 Polyclonal Antibody (Invitrogen #PA5–22124), β-actin (1:250, clone AC15; Sigma Aldrich) and secondary HRP-linked antibodies were purchased from Santa Cruz Biotechnology Inc. For the expression levels of respiratory complexes, the Total OXPHOS Human WB Antibody Cocktail was used (Abcam, Cambridge, MA). Western blots were acquired, using the Azure (C300) imaging system (CCD – camera based).
Ishida C.T., Zhang Y., Bianchetti E., Shu C., Trang N.T., Kleiner G., Sanchez-Quintero M.J., Quinzii C.M., Westhoff M.A., Karpel-Massler G., Prabhu V.V., Allen J.E, & Siegelin M.D. (2018). Metabolic Reprogramming by Dual AKT/ERK Inhibition Through Imipridones Elicits Unique Vulnerabilities in Glioblastoma. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(21), 5392-5406.
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7

Western Blot Protein Analysis

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The cells were harvested in RIPA lysis buffer (EMD Millipore, CA) contain one tablet of Pierce™ protease and phosphatase inhibitor (Thermo Fisher Scientific), and the proteins were purified using a Branson Digital Sonifier homogenizer (Branson Ultrasonics, CT). 20 μg of protein from each sample was separated on NuPAGE 4–12% Bis-Tris protein gels (Thermo Fisher Scientific) and transferred to nitrocellulose membranes (Thermo Fisher Scientific). The protein-bound membranes were blocked with 5% of blotting-grade blocker (Bio-Rad) in PBST for 1 h at room temperature and incubated with a primary antibody (1:1,000 dilution) in 5% of blotting-grade blocker in PBST overnight at 4°C. After washing with PBST buffer, the membranes were incubated with horseradish peroxidase (HRP)-conjugated-secondary antibody for 1 h at room temperature. The membranes were developed with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and exposed on a ChemiDoc Touch imaging system (Bio-Rad) for imaging. The primary antibody used in this study was total OXPHOS human WB antibody cocktail (Abcam, ab110411). The secondary antibodies was HRP-conjugated-goat anti-mouse IgG (SouthernBiotech, 1030-05).
Zhang P., Liu Y., Li C., Stine L.D., Wang P.H., Turnbull M.W., Wu H, & Liu Q. (2023). Ectopic expression of SARS-CoV-2 S and ORF-9B proteins alters metabolic profiles and impairs contractile function in cardiomyocytes. Frontiers in Cell and Developmental Biology, 11, 1110271.
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8

Protein Expression Analysis by Western Blot

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Westerns were performed using the following antibodies α-Tubulin (B-5-1-2, 1:5000, Thermo Scientific); HuR (3A2, 1:4000, Santa Cruz Biotechnology) COX-2 (H-62, 1:1000, Santa Cruz), Total OXPHOS Human WB Antibody Cocktail (ab110411, 1:1000, Abcam), Anti-TOMM20 antibody (ab56783, 1:1000, Abcam), Beta-actin (13E5 1:10,000, Cell Signaling). Membranes were scanned using Odyssey Infrared Imaging System (LI-COR Biosciences) and analyzed with Image Studio Lite Version 5.2.
Schultz C.W., McCarthy G.A., Nerwal T., Nevler A., DuHadaway J.B., McCoy M.D., Jiang W., Brown S.Z., Goetz A., Jain A., Calvert V.S., Vishwakarma V., Wang D., Preet R., Cassel J., Summer R., Shaghaghi H., Pommier Y., Baechler S.A., Pishvaian M.J., Golan T., Yeo C.J., Petricoin E.F., Prendergast G.C., Salvino J., Singh P.K., Dixon D.A, & Brody J.R. (2021). The FDA approved anthelmintic Pyrvinium Pamoate inhibits pancreatic cancer cells in nutrient depleted conditions by targeting the mitochondria. Molecular cancer therapeutics, 20(11), 2166-2176.
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9

Cybrids and OXPHOS Biochemical Assays

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Fibroblast cells were grown in DMEM medium supplemented with glutamax (446 mg/l), 10% fetal calf serum, 50μg/ml uridine and 1mM sodium pyruvate under standard conditions. Cells were fused with 143B rhoo cells as previously described [63 (link)]. Several clones were isolated and the resulting cybrid cells were subsequently expanded. Biochemical assays were performed on isolated mitochondria and/or permeabilized cells. Western blot analysis of a patient’s muscle biopsy sample and Blue Native gel analysis (BNG) of a patient’s fibroblast were performed as previously described [63 (link)]. We used “MitoProfile” antibody mix (Total OXPHOS human WB antibody cocktail, Abcam) for muscle lysates. For fibroblast lysates we used antibody GRIM19 (Abcam), which corresponds to the mitochondrial complex I subunit NDUFA13.
Simon M., Richard E.M., Wang X., Shahzad M., Huang V.H., Qaiser T.A., Potluri P., Mahl S.E., Davila A., Nazli S., Hancock S., Yu M., Gargus J., Chang R., Al-sheqaih N., Newman W.G., Abdenur J., Starr A., Hegde R., Dorn T., Busch A., Park E., Wu J., Schwenzer H., Flierl A., Florentz C., Sissler M., Khan S.N., Li R., Guan M.X., Friedman T.B., Wu D.K., Procaccio V., Riazuddin S., Wallace D.C., Ahmed Z.M., Huang T, & Riazuddin S. (2015). Mutations of Human NARS2, Encoding the Mitochondrial Asparaginyl-tRNA Synthetase, Cause Nonsyndromic Deafness and Leigh Syndrome. PLoS Genetics, 11(3), e1005097.
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10

Profiling OXPHOS Proteins in Subjects via Immunoblotting

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Immunoblotting was performed as described previously.10 Primary antibodies were Anti‐C22orf25 antibody (Abcam, ab87576), GAPDH antibody (FL‐335) (Santa Cruz, sc‐25778), alpha tubulin (Abcam, ab7291), Anti‐VDAC1 (Abcam, ab14734). Oxidative phosphorylation (OXPHOS) proteins were probed using Total OXPHOS Human WB Antibody Cocktail (Abcam, ab110411) in subjects 1, 2.1 2.2, 4, 6. In Family 2 proteins were detected fluorescently using the Total OXPHOS Antibody Cocktail and Anti‐VDAC1/Porin antibody (Abcam, ab154856) using fluorescently labeled secondary antibodies (Licor IRDye 800CW anti‐rabbit, 926‐32211 and Licor IRDYE 680RD anti‐mouse, 925‐68070) and visualized on a Licor Odyssey CLx.
Mingirulli N., Pyle A., Hathazi D., Alston C.L., Kohlschmidt N., O'Grady G., Waddell L., Evesson F., Cooper S.B., Turner C., Duff J., Topf A., Yubero D., Jou C., Nascimento A., Ortez C., García‐Cazorla A., Gross C., O'Callaghan M., Santra S., Preece M.A., Champion M., Korenev S., Chronopoulou E., Anirban M., Pierre G., McArthur D., Thompson K., Navas P., Ribes A., Tort F., Schlüter A., Pujol A., Montero R., Sarquella G., Lochmüller H., Jiménez‐Mallebrera C., Taylor R.W., Artuch R., Kirschner J., Grünert S.C., Roos A, & Horvath R. (2019). Clinical presentation and proteomic signature of patients with TANGO2 mutations. Journal of Inherited Metabolic Disease, 43(2), 297-308.
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