Native PAGE Analysis of OXPHOS Subunits

Native PAGE was carried out as previously reported [51 (link)]. Briefly, 3–13% acrylamide gradient gels were loaded with 60 μg of mitochondrial protein and processed as described. After electrophoresis, proteins were transferred to a PROTAN^® nitrocellulose membrane (Schleicher and Schuell, Bioscience GmbH, Dassel, Germany) at 35 V, overnight, and probed with specific antibodies raised against the following human OXPHOS subunits: NDUFA9, CORE2, COX2, COX5A, and SDHA (from Mitosciences, Eugene, OR, USA). Peroxidase-conjugated anti-mouse IgG was used as a secondary antibody (Molecular Probes, Thermofisher Scientific, Gilligham, UK). The signal was detected with ECL^® plus (Amersham Biosciences, Freiburg, Germany).

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Aye C.C., Hammond D.E., Rodriguez-Cuenca S., Doherty M.K., Whitfield P.D., Phelan M.M., Yang C., Perez-Perez R., Li X., Diaz-Ramos A., Peddinti G., Oresic M., Vidal-Puig A., Zorzano A., Ugalde C, & Mora S. (2023). CBL/CAP Is Essential for Mitochondria Respiration Complex I Assembly and Bioenergetics Efficiency in Muscle Cells. International Journal of Molecular Sciences, 24(4), 3399.

Publication 2023

NDUFA9 CORE2 COX5A Peroxidase-conjugated anti-mouse IgG ECL® plus

Acrylamide Anti igg Antibodies Antibody Cox2 Electrophoresis Gels Human Membrane Mitochondrial protein Molecular probes Mouse Native page Nitrocellulose Peroxidase Proteins Subunits

Corresponding Organization : Universitat de Barcelona

Other organizations : Wellcome/MRC Institute of Metabolic Science, Addenbrooke's Hospital, University of Cambridge, Wellcome Trust, University of the Highlands and Islands, University of Glasgow, Hospital Universitario 12 De Octubre, Centro de Investigación Biomédica en Red, Centre for Biomedical Network Research on Rare Diseases, Institute for Research in Biomedicine, VTT Technical Research Centre of Finland, Åbo Akademi University, University of Turku, Turku Centre for Biotechnology, Örebro University, Wellcome Sanger Institute, Instituto de Salud Carlos III, Centro de Investigación Biomédica en Red Diabetes y Enfermedades Metabólicas Asociadas

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Variable analysis

independent variables

Acrylamide gradient (3–13%)

dependent variables

Expression levels of NDUFA9, CORE2, COX2, COX5A, and SDHA OXPHOS subunits

control variables

Mitochondrial protein load (60 μg)

controls

Positive controls: Not specified
Negative controls: Not specified

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