[
Methods Cell Biol,
1995]
The ultimate goal of subcellular fractionation and biochemical purification is to better understand the relationships between structure and function of proteins and protein assemblies. Examples of such relationships with respect to specific gene products include the formation of stable complexes, elucidation of catalytic activities, and subcellular localization of the organellar and supramolecular levels. The detailed aspects of such relationships are not always readily predictable from genetic or molecular studies of the gene products or from their cellular localization by immunological methods. Subcellular fractionation and biochemical purification are generally prerequisites to experimental analysis of biochemical mechanisms underlying a biological phenomenon. These approaches can mutually enhance and interact with parallel cellular, genetic, and molecular analyses. To achieve such goals, methods for isolating proteins and protein assemblies must preserve both structural integrity and biological activity. Ideally, both objectives should be met; practically, it may be critical to know which of these conditions is true. In general, specific protocols must be designed for the optimal isolation, purification, and characterization of each specific protein of interest. Additionally, one wishes to achieve as high a yield as possible; however, each step in protein purification generally produces some reduction in yield...