Individual mitochondrion characterization: a comparison of classical assays to capillary electrophoresis with laser-induced fluorescence detection.
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.
Strack
A
A
Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Duffy
C F
CF
Malvey
M
M
Arriaga
E A
EA
D000447:Aldehydes / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D006224:Cricetinae; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D007834:Lasers; D051379:Mice; D008928:Mitochondria / Q000737:chemistry* / Q000378:metabolism; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism
D000447:Aldehydes; D005456:Fluorescent Dyes; D011506:Proteins; C111472:mitotracker green FM