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Over several years we have provided evidence that uncoupling protein 1 (UCP1) is present in thymus mitochondria. We have demonstrated the conclusive evidence for the presence of UCP1 in thymus mitochondria and we have been able to demonstrate a GDP-sensitive UCP1-dependent proton leak in non-phosphorylating thymus mitochondria. In this chapter, we show how to detect UCP1 in mitochondria isolated from whole thymus using immunoblotting. We show how to measure GDP-sensitive UCP1-dependent oxygen consumption in non-phosphorylating thymus mitochondria and we show that increased reactive oxygen species production occurs on addition of GDP to non-phosphorylating thymus mitochondria. We conclude that reactive oxygen species production rate can be used as a surrogate for detecting UCP1 catalyzed proton leak activity in thymus mitochondria.

Original publication

DOI

10.1007/978-1-4939-1875-1_11

Type

Journal article

Journal

Methods Mol Biol

Publication Date

2015

Volume

1241

Pages

123 - 135

Keywords

Animals, Biological Transport, Cell Fractionation, Electrophoresis, Polyacrylamide Gel, Female, Guanosine Diphosphate, Hydrogen Peroxide, Immunoblotting, Ion Channels, Male, Mitochondria, Mitochondrial Proteins, Oxygen Consumption, Protons, Rats, Rats, Wistar, Thymus Gland