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  • Measurement of mitochondrial respiratory chain enzymatic activities in Drosophila melanogaster samples.

Measurement of mitochondrial respiratory chain enzymatic activities in Drosophila melanogaster samples.

STAR protocols (2022-04-29)
Michele Brischigliaro, Elena Frigo, Erika Fernandez-Vizarra, Paolo Bernardi, Carlo Viscomi
ABSTRACT

Mitochondrial respiratory chain (MRC) dysfunction is linked to mitochondrial disease as well as other common conditions such as diabetes, neurodegeneration, cancer, and aging. Thus, the evaluation of MRC enzymatic activities is fundamental for diagnostics and research purposes on experimental models. Here, we provide a verified and reliable protocol for mitochondria isolation from various D. melanogaster samples and subsequent measurement of the activity of MRC complexes I-V plus citrate synthase (CS) through UV-VIS spectrophotometry. For complete details on the use and execution of this protocol, please refer to Brischigliaro et al. (2021).

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Pyruvate Kinase from rabbit muscle, Type II, ammonium sulfate suspension, 350-600 units/mg protein
Sigma-Aldrich
HEPES, ≥99.5% (titration)
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, essentially fatty acid free, ≥96% (agarose gel electrophoresis)
Sigma-Aldrich
Ethylenediaminetetraacetic acid disodium salt dihydrate, 98.5-101.5%, BioUltra
Sigma-Aldrich
Antimycin A from Streptomyces sp.
Sigma-Aldrich
Rotenone, ≥95%
Sigma-Aldrich
L-Lactic Dehydrogenase from rabbit muscle, Type II, ammonium sulfate suspension, 800-1,200 units/mg protein
Sigma-Aldrich
Cytochrome c from equine heart, ≥95% based on Mol. Wt. 12,384 basis
Sigma-Aldrich
Oligomycin from Streptomyces diastatochromogenes, ≥90% total oligomycins basis (HPLC)