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Protocol for the Analysis of Yeast and Human Mitochondrial Respiratory Chain Complexes and Supercomplexes by Blue Native Electrophoresis.

STAR protocols (2020-10-01)
Alba Timón-Gómez, Rafael Pérez-Pérez, Eva Nyvltova, Cristina Ugalde, Flavia Fontanesi, Antoni Barrientos
ABSTRACT

By using negatively charged Coomassie brilliant blue G-250 dye to induce a charge shift on proteins, blue native polyacrylamide gel electrophoresis (BN-PAGE) allows resolution of enzymatically active multiprotein complexes extracted from cellular or subcellular lysates while retaining their native conformation. BN-PAGE was first developed to analyze the size, composition, and relative abundance of the complexes and supercomplexes that form the mitochondrial respiratory chain and OXPHOS system. Here, we present a detailed protocol of BN-PAGE to obtain robust and reproducible results. For complete details on the use and execution of this protocol, please refer to Lobo-Jarne et al. (2018) and Timón-Gómez et al. (2020).

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Trizma® base, Primary Standard and Buffer, ≥99.9% (titration), crystalline
Sigma-Aldrich
Trypsin-EDTA solution, 0.25%, sterile-filtered, BioReagent, suitable for cell culture, 2.5 g porcine trypsin and 0.2 g EDTA • 4Na per liter of Hanks′ Balanced Salt Solution with phenol red
Sigma-Aldrich
Protease Inhibitor Cocktail, for use with mammalian cell and tissue extracts, DMSO solution
Sigma-Aldrich
Glycine, ReagentPlus®, ≥99% (HPLC)
Sigma-Aldrich
Sodium chloride, for molecular biology, DNase, RNase, and protease, none detected, ≥99% (titration)
Sigma-Aldrich
Digitonin, Used as non-ionic detergent
Millipore
Digitonin, High Purity - CAS 11024-24-1 - Calbiochem, CAS 11024-24-1 is a non-ionic detergent commonly used to solubilize membrane-bound proteins.
Sigma-Aldrich
6-Aminocaproic acid, ≥99% (titration), powder
Sigma-Aldrich
Uridine, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
3,3′-Diaminobenzidine tetrahydrochloride, tablet, 10 mg substrate per tablet
Sigma-Aldrich
Cytochrome c from bovine heart, ≥95% based on Mol. Wt. 12,327 basis
Sigma-Aldrich
Apoferritin from equine spleen
Sigma-Aldrich
Nicotinic acid adenine dinucleotide sodium salt, ≥98%
Supelco
(±)-cis-4,4′-Dimethylaminorex solution, 1.0 mg/mL (1% 1M HCl in Methanol), ampule of 1 mL, certified reference material, Cerilliant®