Subcellular fractionation by differential centrifugation for mitochondrial studies

In addition to fluorescence microscopy, the subcellular fractionation of eukaryotic cells remains one of the central methods for the basic characterization of proteins. Here we describe an optimized procedure for the subcellular fractionation of yeast cells, specifically for mitochondrial studies. Major recommendations are to separate the fractions immediately after each centrifugation step, to carefully discard a significant part of the supernatant fractions which is in the direct vicinity to the pellets and, in addition, to perform an extra homogenization step of the post nuclear supernatant fraction. These principles help to collect supernatant fractions with less cross-contaminations from the corresponding pellets. These approaches are scalable and adaptable for the fractionation of other cell types and are also useful for the characterization of other organelles.