A method has been developed that uses capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF) for measuring protein abundance in individual mitochondria collected from a discontinuous density gradient and labeled with Mitotracker Green. From these measurements we determined the distribution of protein content per mitochondrion and the relative abundance of mitochondrial proteins in density gradient fractions. In addition, this method is useful for counting mitochondria and, as a consequence, determining the number of mitochondria per unit volume or estimate mitochondria copy number per cell. It was determined that mitochondria accumulate in two interfaces defined by consecutive layers of 35% Metrizamide, 17% Metrizamide, and 6% Percoll. The presence of mitochondria in these interfaces was also confirmed using a modified Lowry assay that prevents interference from Metrizamide and Percoll and determines total protein content, and a succinate dehydrogenase assay that uses dichloroindophenol as an electron acceptor and that specifically indicates abundance of mitochondria. The CE-LIF analysis of mitochondrial properties, based on the individual mitochondrial determinations, has a wider scope than the average values determined by enzymatic or bulk protein assays.
Bibliographical noteFunding Information:
A.S. was supported by the LANDO-NSF Summer Program; traveling funds for C.D. were provided by the International Union of Biochemistry and Molecular Biology Trust Fund. E.A. thanks the graduate school for a grant-in-aid award. This research team is grateful to Dr. Wei-Shou Hu for providing the initial supply of cultured cells, and Dr. Sally Palm and Ms. Takia Washington for maintaining the cell culture room.