Abstract
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.
Highlights
The purity of the nuclear fraction was assessed by staining for specific markers of the cell membrane (Na/K ATPase), endoplasmic reticulum (ER) (ERp-29), mitochondria, and cytosol (GAPDH)
Aration of nuclei from ER and mitochondria, while the addition of the washing step reWe show that the concentration detergent in a our washing sulted in a significant increase in the qualityofofnon-ionic fractionation
It should be noted that other non-ionic detergents similar to NP-40
Summary
Dozens of studies have demonstrated the importance of nuclear import of proteins for transcription induction, while their export to the cytoplasm decreases the transcription of target genes [1]. Apoptosis is one of the physiological processes characterized by alterations in the localization of multiple proteins, including various examples of translocation of apoptotic regulators between different intracellular compartments [2]. Upon apoptosis induction, cytochrome c translocates from the mitochondria to cytosol, while AIF, endonuclease G (EndoG), and HtrA2/Omi are released from the intermembrane space of the mitochondria but translocate into the nucleus [3,4]. Caspases themselves, which are the main players in apoptosis induction and execution, have been shown to translocate to the nucleus and cleave many of their nuclear substrates [6,7]
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