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, relat...

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Bibliographic Details
Published in:The Journal of cell biology 1965-11, Vol.27 (2), p.365-378
Main Authors: Edelman, Jean C., Edelman, P. Michael, Knigge, Karl M., Schwartz, Irving L.
Format: Article
Language:English
Subjects:
Adenosine Triphosphatases
Adenosine Triphosphate
Animals
BASIC BIOLOGICAL SCIENCES
Cell Nucleus
Centrifugation
Cytochromes
DNA
Electron Transport Complex IV
Filtration
Histological Techniques
Homogenization
In Vitro Techniques
Liver
Microscopy, Electron
Microscopy, Phase-Contrast
Mitochondria
Muscles - cytology
Myofibrils
Nuclear membrane
Nucleoproteins
Oxidases
Rats
RNA
Skeletal muscle
Sodium Chloride
Striated muscle
Subcellular Fractions
Sucrose
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Summary: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.
ISSN:0021-9525
1540-8140
DOI:10.1083/jcb.27.2.365