Subcellular Fractionation to Demonstrate Activation of Intrinsic Apoptotic Pathway.

Within the cell, proteins are segregated into different organelles depending on their function and activation status. In response to stimulus, posttranslational modifications or loss of organelle membrane integrity lead to the movement of proteins from one compartment to another. This movement of proteins or protein translocation, exerts a significant effect on protein function. This is clearly demonstrated in the context of apoptosis wherein the cytoplasmic translocation of the mitochondrial resident protein, cytochrome C, initiates the activation of the intrinsic arm of the apoptotic pathway. Experimentally, protein translocation can be demonstrated by subcellular fractionation and subsequent western blot analysis of the isolated fractions. This chapter describes the step-by-step procedure in obtaining mitochondrial and cytoplasmic fractions from cell pellets and determining their purity and integrity.