Subcellular fractionation, electromigration analysis and mapping of organelles

Subcellular fractionation has provided the means required to analyze the co mposition and properties of purified cellular elements. In particular, subc ellular fractionation has helped to define membrane boundaries and became n ecessary for the development of cell-free assays that reconstitute complica ted cellular processes. Although cell fractionation techniques have improve d over the last decades the purification of organelles to homogeneity is st ill a barely accessible goal in cell biology. In this article, we will firs t briefly review the basic principles of subcellular fractionation, and the establishment of different organelle fractions by density centrifugation, using tissue culture cells as a paradigm. Then we will discuss some of the intrinsic problems and will compare gradient purification of cellular extra cts with electromigration analysis. Finally, we will describe alternative a pproaches, such as immunoisolation and flow cytometry to purify organelles from tissue culture cells. (C) 1999 Elsevier Science B.V. All rights reserv ed.