Purification of Saccharomyces cerevisiae mitochondria by zone electrophoresis in a free flow device.
This chapter describes the isolation of yeast mitochondria by differential centrifugation followed by mitochondrial purification through zone electrophoresis (ZE) using a free flow device (FFE). Starting from a yeast colony, cultures are grown under respiratory conditions to logarithmic phase. Cells are collected, their cell walls enzymatically disintegrated and the resulting spheroplasts are homogenized. Mitochondria are pre-fractionated from this homogenate by differential centrifugation. With the focus on further purification, pre-fractionated mitochondria are subjected to ZE-FFE. In ZE-FFE, mitochondria are transported with the buffer flow through the separation chamber and purified from contaminants by specific deflection through a perpendicularly oriented electric field. The purified mitochondria can be collected by centrifugation and used for further experiments and analysis such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, 2-DE or electron microscopy.
Zischka
Hans
H
GSF-National Research Center for Environment and Health, Institute of Toxicology, Munich-Neuherberg, Germany.
Kinkl
Norbert
N
Braun
Ralf J
RJ
Ueffing
Marius
M
D000251:Adenosine Triphosphatases / Q000302:isolation & purification; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification; D002498:Centrifugation / Q000379:methods; D014450:Electron Transport Complex III / Q000302:isolation & purification; D003576:Electron Transport Complex IV / Q000302:isolation & purification; D006736:Horses; D015151:Immunoblotting; D008565:Membrane Proteins / Q000302:isolation & purification; D008928:Mitochondria / Q000737:chemistry* / Q000648:ultrastructure*; D025261:Mitochondrial Proton-Translocating ATPases; D009154:Mutation; D010085:Oxidative Phosphorylation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000648:ultrastructure*; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification; D014451:Ubiquinone / Q000031:analogs & derivatives
D002352:Carrier Proteins; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D014451:Ubiquinone; D003576:Electron Transport Complex IV; D000251:Adenosine Triphosphatases; D025261:Mitochondrial Proton-Translocating ATPases; D014450:Electron Transport Complex III; C020439:oligomycin sensitivity-conferring protein; C003741:ubiquinol