High Throughput Cell Fractionation

The movement of pro-apoptotic factors of the Bcl2-family from the cytosol to the mitochondria and the consequent permeabilization of the mitochondrial outer membrane to release cytochrome c and Smac from the mitochondrial intermembrane space into the cytosol or AIF to the nucleus are now well characterized steps in programmed cell death (Figure 1). However, there are concurrent movements of other proteins, including kinases and transcription factors (e.g. p53), to and from the organelle to both signal and modulate apoptosis. The identification and quantification of these protein movements between cellular compartments is necessary to fully understand and to differentiate between different pathways of cell death. Current methodology of monitoring such movements mostly relies on difficult-to-quantify immunocytochemical approaches or on biochemical cell fractionation that involves mechanical disruption of cells followed by a complicated centrifugation scheme.