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The Journal of Cell Biology, Vol 27, 365-378, Copyright © 1965 by Rockefeller University Press

ARTICLE

ISOLATION OF SKELETAL MUSCLE NUCLEI

Jean C. Edelman 1, P. Michael Edelman 1, Karl M. Knigge 1, and Irving L. Schwartz 1

1 From the Departments of Physiology and Anatomy, University of Cincinnati College of Medicine, Cincinnati, Ohio, and The Medical Research Center, Brookhaven National Laboratory, Upton, New York.

Dr. Knigge's present address is the Department of Anatomy, University of Rochester School of Medicine and Dentistry, Rochester, New York

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.

Submitted on June 14, 1965


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