期刊
ANALYTICAL BIOCHEMISTRY
卷 293, 期 2, 页码 269-276出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1006/abio.2001.5139
关键词
mitochondrial membrane potential; fluorescent probe; high-throughput screening; uncoupling protein 1
A fluorometric assay for mitochondrial membrane potential in permeabilized yeast cells has been developed. This method involves permeabilizing the plasma membrane and measuring the distribution of a mitochondrial membrane potential sensitive probe 3,3 ' -dipropylthiadicarbocyanine iodide (DISC3(5); DiSC(3)), In permeabilized cells, DISC, fluorescence decreased when introduced into energized mitochondria and increased three- to sixfold when the mitochondrial membrane potential was dissipated by the chemical uncoupler carbonylcyanide m-chlorophenyl hydrazone. Plasma membrane potential was abolished by permeabilization, as shown by a lack of polarization of the plasma membrane induced by K+ and glucose. Uncoupling protein 1 (UCP1), a mitochondrial H+ transporter, was used as a model for method validation. The fluorescence intensity responded vigorously to specific modulators in UCP1-expressing cells. This method has been adapted as a high-throughput assay to screen for modulators of mitochondrial membrane potential. (C) 2001 Academic Press.
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