ISOLATION OF SKELETAL MUSCLE NUCLEI

A method employing aqueous media for isolation of nuclei from rat skeletal muscle is described. The technique involves (a) mincing and then homogenizing in a 0.32 M sucrose-salt solution with a Potter-Elvehjem type homogenizer using a Delrin (an acetal resin) pestle and a carefully controlled, rela...

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Main Authors:	Edelman, Jean C., Edelman, P. Michael, Knigge, Karl M., Schwartz, Irving L.
Format:	Online
Language:	English
Published:	The Rockefeller University Press 1965
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2106724/

id	pubmed-2106724
recordtype	oai_dc
spelling	pubmed-21067242008-05-01 ISOLATION OF SKELETAL MUSCLE NUCLEI Edelman, Jean C. Edelman, P. Michael Knigge, Karl M. Schwartz, Irving L. Article A method employing aqueous media for isolation of nuclei from rat skeletal muscle is described. The technique involves (a) mincing and then homogenizing in a 0.32 M sucrose-salt solution with a Potter-Elvehjem type homogenizer using a Delrin (an acetal resin) pestle and a carefully controlled, relatively large pestle-to-glass clearance, (b) filtering through fiberglass and stainless steel screens of predetermined mesh size to remove myofibrils and connective tissue, and (c) centrifuging in a 2.15 M sucrose-salt solution containing 0.7 mM ATP. Electron and phase-contrast microscopic observations show that the nuclei are intact, unencumbered by cytoplasmic tags, and possess well preserved distinct nucleoli, nucleoplasm, and nuclear membranes. Cytoplasmic contamination is minimal and mainly mitochondrial. Chemical assays of the nuclear fraction show that the DNA/protein and RNA/DNA ratios are comparable to those obtained in other tissues. These ratios, as well as the low specific activity obtained for cytochrome c oxidase and the virtual absence of myofibrillar ATPase, indicate a high degree of purity with minimal mitochondrial and myofibrillar contamination. The steps comprising the technique and the reasons for their selection are discussed. The Rockefeller University Press 1965-11-01 /pmc/articles/PMC2106724/ /pubmed/4287141 Text en This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Edelman, Jean C. Edelman, P. Michael Knigge, Karl M. Schwartz, Irving L.
spellingShingle	Edelman, Jean C. Edelman, P. Michael Knigge, Karl M. Schwartz, Irving L. ISOLATION OF SKELETAL MUSCLE NUCLEI
author_facet	Edelman, Jean C. Edelman, P. Michael Knigge, Karl M. Schwartz, Irving L.
author_sort	Edelman, Jean C.
title	ISOLATION OF SKELETAL MUSCLE NUCLEI
title_short	ISOLATION OF SKELETAL MUSCLE NUCLEI
title_full	ISOLATION OF SKELETAL MUSCLE NUCLEI
title_fullStr	ISOLATION OF SKELETAL MUSCLE NUCLEI
title_full_unstemmed	ISOLATION OF SKELETAL MUSCLE NUCLEI
title_sort	isolation of skeletal muscle nuclei
description	A method employing aqueous media for isolation of nuclei from rat skeletal muscle is described. The technique involves (a) mincing and then homogenizing in a 0.32 M sucrose-salt solution with a Potter-Elvehjem type homogenizer using a Delrin (an acetal resin) pestle and a carefully controlled, relatively large pestle-to-glass clearance, (b) filtering through fiberglass and stainless steel screens of predetermined mesh size to remove myofibrils and connective tissue, and (c) centrifuging in a 2.15 M sucrose-salt solution containing 0.7 mM ATP. Electron and phase-contrast microscopic observations show that the nuclei are intact, unencumbered by cytoplasmic tags, and possess well preserved distinct nucleoli, nucleoplasm, and nuclear membranes. Cytoplasmic contamination is minimal and mainly mitochondrial. Chemical assays of the nuclear fraction show that the DNA/protein and RNA/DNA ratios are comparable to those obtained in other tissues. These ratios, as well as the low specific activity obtained for cytochrome c oxidase and the virtual absence of myofibrillar ATPase, indicate a high degree of purity with minimal mitochondrial and myofibrillar contamination. The steps comprising the technique and the reasons for their selection are discussed.
publisher	The Rockefeller University Press
publishDate	1965
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2106724/
_version_	1611409084106407936

ISOLATION OF SKELETAL MUSCLE NUCLEI

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