Simple and Efficient Protocol for Subcellular Fractionation of Normal and Apoptotic Cells

Subcellular fractionation approaches remain an indispensable tool among a large number of biochemical methods to facilitate the study of specific intracellular events and characterization of protein functions. During apoptosis, the best-known form of programmed cell death, numerous proteins are translocated into and from the nucleus. Therefore, suitable biochemical techniques for the subcellular fractionation of apoptotic cells are required. However, apoptotic bodies and cell fragments might contaminate the fractions upon using the standard protocols. Here, we compared different nucleus/cytoplasm fractionation methods and selected the best-suited approach for the separation of nuclear and cytoplasmic contents. The described methodology is based on stepwise lysis of cells and washing of the resulting nuclei using non-ionic detergents, such as NP-40. Next, we validated this approach for fractionation of cells treated with various apoptotic stimuli. Finally, we demonstrated that nuclear fraction could be further subdivided into nucleosolic and insoluble subfractions, which is crucial for the isolation and functional studies of various proteins. Altogether, we developed a method for simple and efficient nucleus/cytoplasm fractionation of both normal and apoptotic cells.

Automated summary

Subcellular fractionation remains essential for studying specific intracellular events and characterizing protein functions, especially during apoptosis. By comparing different fractionation methods, this study developed a simple and efficient approach for nucleus/cytoplasm separation, validated for both normal and apoptotic cells. The method involves stepwise cell lysis with NP-40 to isolate nuclei, further subdivided into soluble and insoluble fractions for protein studies.