Total oxphos antibody cocktail

Manufactured by Abcam

Sourced in United States, United Kingdom

The Total OXPHOS antibody cocktail is a collection of primary antibodies that can detect the five essential subunits of the oxidative phosphorylation (OXPHOS) complexes in mammalian cells. This product provides a convenient way to measure the relative abundance of OXPHOS complexes in various samples.

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Lab products found in correlation

Pgc 1α, by Abcam (2 mentions) Vinculin, by Merck Group (2 mentions) Tom20, by Santa Cruz Biotechnology (2 mentions) Serca2, by Thermo Fisher Scientific (2 mentions) Flag tag (flag), by Cell Signaling Technology (2 mentions) Ckmt2, by Abcam (2 mentions) Serca1, by Abcam (2 mentions) Hrp conjugated anti mouse igg, by EASYBIO (2 mentions) Akt 9272, by Cell Signaling Technology (1 mentions) Mab6158, by R&D Systems (1 mentions) β tubulin, by R&D Systems (1 mentions) P smad1 5 9, by Cell Signaling Technology (1 mentions) Phospho akt ser473, by Cell Signaling Technology (1 mentions) Ripa buffer, by Thermo Fisher Scientific (1 mentions) Mitochondria isolation kit for tissue, by Thermo Fisher Scientific (1 mentions) Pvdf membrane, by Bio-Rad (1 mentions) Anti gapdh, by Cell Signaling Technology (1 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions) Anti plin2, by Cell Signaling Technology (1 mentions) Mitochondria cytosol fractionation kit, by Abcam (1 mentions)

Spelling variants of this product

OXPHOS cocktail (40 citations) Total OXPHOS Human WB Antibody Cocktail (29 citations) MitoProfile Total OXPHOS Rodent WB Antibody Cocktail (26 citations) Total OXPHOS (24 citations) OXPHOS antibody cocktail (23 citations) Total OXPHOS cocktail (18 citations) MitoProfile total OXPHOS antibody cocktail (9 citations) Total OXPHOS human antibody cocktail (5 citations) Mouse anti-total OXPHOS antibody cocktail (2 citations) Total OXPHOS Blue Native WB Antibody Cocktail (2 citations)

21 protocols using total oxphos antibody cocktail

Immunoblotting Antibody Quantification Protocol

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Immunoblotting was performed as described [14 (link)] with the following commercial primary antibodies: SMAD 1/5/9 (ab66737, Abcam, Cambridge, UK), total OXPHOS Antibody Cocktail (ab 110413, Abcam, Cambridge, UK), UCP1 (MAB6158, R&D System), β-tubulin (#2128), pSMAD 1/5/9 (#13820), Akt (#9272) and phospho-Akt (ser 473) (#9271) (all from Cell Signalling Technology, Danvers, MA, USA) and phospho-Akt (thr308) (#9275S BioLabs). Quantifications were performed by normalisation against loading controls.

Khatib Shahidi R., M. Hoffmann J., Hedjazifar S., Bonnet L., K. Baboota R., Heasman S., Church C., Elias I., Bosch F., Boucher J., Hammarstedt A, & Smith U. (2021). Adult mice are unresponsive to AAV8-Gremlin1 gene therapy targeting the liver. PLoS ONE, 16(2), e0247300.

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OXPHOS and Mitochondrial Protein Analysis

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The following antibodies were used in this study: total OXPHOS antibody cocktail (Abcam, ab110413); Ckmt2 (Abcam, ab55963); Flag (Cell Signaling, 14793); Pgc1-α (Abcam, ab54481); Tom-20 (SantaCruz, sc-11415); Serca1 (Abcam, ab109899); Serca2 (Invitrogen, MA3-919); Ucp1 (Abcam, ab 10983); vinculin (Sigma, V9131).

Pollard A.E., Martins L., Muckett P.J., Khadayate S., Bornot A., Clausen M., Admyre T., Bjursell M., Fiadeiro R., Wilson L., Whilding C., Kotiadis V.N., Duchen M.R., Sutton D., Penfold L., Sardini A., Bohlooly-Y M., Smith D.M., Read J.A., Snowden M.A., Woods A, & Carling D. (2019). AMPK activation protects against diet induced obesity through Ucp1-independent thermogenesis in subcutaneous white adipose tissue. Nature metabolism, 1(3), 340-349.

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Hippocampal Protein Expression Analysis

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Hippocampal brain and cellular extracts were homogenized in radioimmunoprecipitation assay lysis buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1 SDS, 1 mM EDTA, 10% glycerol, and protease inhibitors] and resolved by SDS–polyacrylamide gel electrophoresis in 8 to 15% polyacrylamide gels. The concentration of the proteins was determined by Bradford protein assay using bovine serum albumin as standard and analyzed by Western blotting with specific antibodies.
Antibodies used were as follows: rabbit-MPC1 (Sigma-Aldrich, HPA045119), mouse-MPC2 (Merck, MABS1914-25UG), goat-voltage dependent anion channel protein (VDAC) (Santa Cruz Biotechnology, sc-8829), mouse-HSP70 (Invitrogen, MA3-028), total OXPHOS antibody cocktail (Abcam, MS604-300), anti–immunoglobulin G (IgG)–rabbit–horseradish peroxidase (HRP) (Dako, P0217), anti–IgG-mouse-HRP (Dako, P0447), and anti–IgG-Goat-HRP (Santa Cruz Biotechnology, sc-2304).

Petrelli F., Scandella V., Montessuit S., Zamboni N., Martinou J.C, & Knobloch M. (2023). Mitochondrial pyruvate metabolism regulates the activation of quiescent adult neural stem cells. Science Advances, 9(9), eadd5220.

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OXPHOS and Mitochondrial Protein Analysis

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Protein Profiling of C2C12 Cells and Skeletal Muscle

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Total protein was extracted from C2C12 cells and skeletal muscle tissue using RIPA buffer (Thermo Fisher Scientific) and the mitochondrial fraction was prepared using a mitochondria isolation kit for tissue (Thermo Fisher Scientific) following the manufacturer’s protocol. Protein concentration was measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Protein samples were loaded and separated on a 12% SDS PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). Non-specific sites were blocked with 5% skim milk in TBST and the membrane was incubated with primary antibodies overnight at 4° C, followed by incubation with HRP-conjugated secondary antibodies for 1 h at room temperature. Antibodies specific to atrogin-1, MuRF1, NCoR1, PGC1α/β, Fkbp5, and PLIN2, as well as a total OXPHOS antibody cocktail were obtained from Abcam (Cambridge, UK). Total and MHC subtype antibodies were supplied by DSHB (IA, USA). Anti-PLIN2 and anti-GAPDH were purchased from Cell Signaling Technologies (MA, USA).

Lee H., Kim Y.I., Nirmala F.S., Kim J.S., Seo H.D., Ha T.Y., Jang Y.J., Jung C.H, & Ahn J. (2021). MiR-141-3p promotes mitochondrial dysfunction in ovariectomy-induced sarcopenia via targeting Fkbp5 and Fibin. Aging (Albany NY), 13(4), 4881-4894.

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Mitochondrial Dysfunction in Choriocarcinoma

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BeWo choriocarcinoma cells were incubated in the presence or absence of 5 mM β-alanine for 72 h before isolation of mitochondria using a mitochondria/cytosol fractionation kit (Abcam). Western blot analysis of subunits in mitochondrial electron transport chain (ETC) complex proteins I–V was then performed using a Total OXPHOS antibody cocktail (Abcam, 1:200 dilution). Nitrocellulose membranes were stripped and re-probed for VDAC (Cell signalling, 1:1,000 dilution). Developed film was scanned and the mean signal intensity of the immunoreactive species determined using Image J software. To account for any variability in sample loading, signal intensity of complex proteins I-V in each sample was normalised to the corresponding VDAC signal intensity.
Mitochondrial morphology and ROS in BeWo cells were assessed using MitoTracker® and CellRox® fluorescent probes respectively. To study the effect of oxidative stress following β-alanine-mediated intracellular taurine depletion, BeWo cells were treated for 1 h with 1 mM H₂O₂ prior to immunofluorescence.

Desforges M., Whittaker H., Farmer E., Sibley C.P, & Greenwood S.L. (2015). Effects of Taurine Depletion on Human Placental Syncytiotrophoblast Renewal and Susceptibility to Oxidative Stress. Advances in experimental medicine and biology, 803, 63-73.

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Immunoblot Analysis of Mitochondrial OXPHOS

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Preparation of tissue lysate was carried out as described previously [93 (link)]. From this, 10 μg protein was loaded into 4–20% gradient gels (Mini-PROTEAN® TGX™ Precast Protein Gels, 15-well, 15 μl #4561096, BioRAD) alongside a protein ladder (Precision plus protein dual color standards #1610374, BioRAD). The gel was transferred to nitrocellulose membrane, and this was stained in Ponceau S. The membrane was blocked in 5% Skimmed milk-TBS-T for 1 h prior to primary antibody incubation (Total OXPHOS antibody cocktail, ab110412, Abcam, RRID:AB_2847807; 1:500 in 1% Milk TBS-T) overnight at 4 °C. The membrane was incubated with secondary antibody (Rabbit anti-Mouse IgG HRP, #61-6520, Invitrogen; RRID:AB_2533933 1:5000 in TBS-T)) for 1 h at room temperature, before ECL detection (Milipore) and imaging using iBright 1500 (ThermoFisher Scientific). Band density was quantified using Image J software [92 (link)]. The original immunoblot image is presented in Additional File 1: Figure S8H.

O’Brien K.A., McNally B.D., Sowton A.P., Murgia A., Armitage J., Thomas L.W., Krause F.N., Maddalena L.A., Francis I., Kavanagh S., Williams D.P., Ashcroft M., Griffin J.L., Lyon J.J, & Murray A.J. (2021). Enhanced hepatic respiratory capacity and altered lipid metabolism support metabolic homeostasis during short-term hypoxic stress. BMC Biology, 19, 265.

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Western Blot Analysis of Myocardial Proteins

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Western blot analysis was carried out as described previously30 (link). In brief, proteins of myocardial tissue and H9c2 cardiomyoblasts were prepared and separated on SDS-PAGE gels. Then, they were transferred to polyvinylidene difluoride membrane (Millipore, USA) and incubated overnight (4 °C) with p-AMPK, AMPK, PGC-1α, Bcl-2, Bax, caspase-3 and cleaved caspase-3 antibodies (Cell Signaling Technology, MA, USA, 1:1000 dilution), SIRT3, SOD2, NRF1, TFAM, cytochrome c and β-actin antibodies (Santa Cruz, CA, USA, 1:500 dilution) and total OXPHOS antibody cocktail (Abcam biotechnology, Cambridge, UK, 1:1000 dilution). Then, the membranes were washed and probed with the secondary antibodies for 2 hours (37 °C). Finally, the positive bands were scanned with ChemiDocXRS and analyzed with Imagelab software system (Bio-Rad, CA, USA).

Yu L., Gong B., Duan W., Fan C., Zhang J., Li Z., Xue X., Xu Y., Meng D., Li B., Zhang M., Bin Z.h.a.n.g., Jin Z., Yu S., Yang Y, & Wang H. (2017). Melatonin ameliorates myocardial ischemia/reperfusion injury in type 1 diabetic rats by preserving mitochondrial function: role of AMPK-PGC-1α-SIRT3 signaling. Scientific Reports, 7, 41337.

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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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Mitochondrial Protein Analysis in Megakaryocytes

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The megakaryocytes from different treatments (0, 10, 50 and 100 ng/mL, 12 d) were transferred to a glass homogenizer and ground in an ice bath for 35 min. Then, the mitochondrial proteins were prepared using a mitochondrial protein extraction kit according to the manufacturer’s instruction (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). After being quantitatively analyzed using a BCA protein assay kit (Beyotime, China), the consistent amount of the protein sample from each group was submitted to Western blot assay. The primary antibody was the total OXPHOS antibody cocktail (1:1000, ab110411, Abcam, USA), and the second antibody was the HRP-conjugated anti-mouse IgG (1:500, EasyBio, China). The quality of this assay was reversely confirmed by stable expressions of the test biomarkers, including mitochondrial respiratory chain complex II, III and V.

Jin X., Yu H., Wang B., Sun Z., Zhang Z., Liu Q.S., Zheng Y., Zhou Q, & Jiang G. (2021). Airborne particulate matters induce thrombopoiesis from megakaryocytes through regulating mitochondrial oxidative phosphorylation. Particle and Fibre Toxicology, 18, 19.

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