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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 tran...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8069826/ https://www.ncbi.nlm.nih.gov/pubmed/33918601 http://dx.doi.org/10.3390/cells10040852 |
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author | Senichkin, Viacheslav V. Prokhorova, Evgeniia A. Zhivotovsky, Boris Kopeina, Gelina S. |
author_facet | Senichkin, Viacheslav V. Prokhorova, Evgeniia A. Zhivotovsky, Boris Kopeina, Gelina S. |
author_sort | Senichkin, Viacheslav V. |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-8069826 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-80698262021-04-26 Simple and Efficient Protocol for Subcellular Fractionation of Normal and Apoptotic Cells Senichkin, Viacheslav V. Prokhorova, Evgeniia A. Zhivotovsky, Boris Kopeina, Gelina S. Cells Article 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. MDPI 2021-04-09 /pmc/articles/PMC8069826/ /pubmed/33918601 http://dx.doi.org/10.3390/cells10040852 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Senichkin, Viacheslav V. Prokhorova, Evgeniia A. Zhivotovsky, Boris Kopeina, Gelina S. Simple and Efficient Protocol for Subcellular Fractionation of Normal and Apoptotic Cells |
title | Simple and Efficient Protocol for Subcellular Fractionation of Normal and Apoptotic Cells |
title_full | Simple and Efficient Protocol for Subcellular Fractionation of Normal and Apoptotic Cells |
title_fullStr | Simple and Efficient Protocol for Subcellular Fractionation of Normal and Apoptotic Cells |
title_full_unstemmed | Simple and Efficient Protocol for Subcellular Fractionation of Normal and Apoptotic Cells |
title_short | Simple and Efficient Protocol for Subcellular Fractionation of Normal and Apoptotic Cells |
title_sort | simple and efficient protocol for subcellular fractionation of normal and apoptotic cells |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8069826/ https://www.ncbi.nlm.nih.gov/pubmed/33918601 http://dx.doi.org/10.3390/cells10040852 |
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