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. 1965 Nov 1;27(2):365–378. doi: 10.1083/jcb.27.2.365

ISOLATION OF SKELETAL MUSCLE NUCLEI

Jean C Edelman 1, P Michael Edelman 1, Karl M Knigge 1, Irving L Schwartz 1
PMCID: PMC2106724  PMID: 4287141

Abstract

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. ALLFREY V. G., MIRSKY A. E., OSAWA S. Protein synthesis in isolated cell nuclei. J Gen Physiol. 1957 Jan 20;40(3):451–490. doi: 10.1085/jgp.40.3.451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. ALLFREY V., STERN H., MIRSKY A. E., SAETREN H. The isolation of cell nuclei in non-aqueous media. J Gen Physiol. 1952 Jan;35(3):529–554. doi: 10.1085/jgp.35.3.529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. CHAUVEAU J., MOULE Y., ROUILLER C. Technique d'isolement des noyaux cellulaires basée sur leur densité. Bull Soc Chim Biol (Paris) 1957;39(12):1521–1533. [PubMed] [Google Scholar]
  4. COOPERSTEIN S. J., LAZAROW A. A microspectrophotometric method for the determination of cytochrome oxidase. J Biol Chem. 1951 Apr;189(2):665–670. [PubMed] [Google Scholar]
  5. DOUNCE A. L., WITTER R. F., MONTY K. J., PATE S., COTTONE M. A. A method for isolating intact mitochondria and nuclei from the same homogenate, and the influence of mitochondrial destruction on the properties of cell nuclei. J Biophys Biochem Cytol. 1955 Mar;1(2):139–153. doi: 10.1083/jcb.1.2.139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. FALZONE J. A., BARROWS C. H., YIENGST M. J. Fractionation of rat liver nuclei into diploid and tetraploid DNA classes by continuous density gradient sedimentation. Exp Cell Res. 1962 Mar;26:552–561. doi: 10.1016/0014-4827(62)90160-x. [DOI] [PubMed] [Google Scholar]
  7. FISCHER F., SIEBERT G., ADLOFF E. [Characterization of 2 adenosine triphosphatases in hog kidney cell nucleus]. Biochem Z. 1959;332:131–150. [PubMed] [Google Scholar]
  8. GURR M. I., FINEAN J. B., HAWTHORNE J. N. THE PHOSPHOLIPIDS OF LIVER-CELL FRACTIONS. I. THE PHOSPHOLIPID COMPOSITION OF THE LIVER-CELL NUCLEUS. Biochim Biophys Acta. 1963 Aug 27;70:406–416. doi: 10.1016/0006-3002(63)90770-4. [DOI] [PubMed] [Google Scholar]
  9. HOGEBOOM G. H., SCHNEIDER W. C., STRIEBICH M. J. Cytochemical studies. V. On the isolation and biochemical properties of liver cell nuclei. J Biol Chem. 1952 May;196(1):111–120. [PubMed] [Google Scholar]
  10. HYMER W. C., KUFF E. L. ISOLATION OF NUCLEI FROM MAMMALIAN TISSUES THROUGH THE USE OF TRITON X-100. J Histochem Cytochem. 1964 May;12:359–363. doi: 10.1177/12.5.359. [DOI] [PubMed] [Google Scholar]
  11. LANG K., SIEBERT G. Untersuchungen über Stoffwechselvorgänge in isolierten Zellkernen. 5. Uber Energielieferung durch Glykolyse und ATP-Spaltung sowie über das Vorkommen von Dehydrasen in isolierten Zellkernen. Biochem Z. 1951 Dec;322(3):196–204. [PubMed] [Google Scholar]
  12. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  13. MAGGIO R., SIEKEVITZ P., PALADE G. E. STUDIES ON ISOLATED NUCLEI. I. ISOLATION AND CHEMICAL CHARACTERIZATION OF A NUCLEAR FRACTION FROM GUINEA PIG LIVER. J Cell Biol. 1963 Aug;18:267–291. doi: 10.1083/jcb.18.2.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. NIEHAUS W. G., Jr, BARNUM C. P. CHARACTERIZATION OF NUCLEI FROM REGENERATING RAT LIVER ON THE BASIS OF INCORPORATION OF RADIOISOTOPE INTO DEOXYRIBONUCLEIC ACID IN VIVO. J Biol Chem. 1964 Apr;239:1198–1201. [PubMed] [Google Scholar]
  15. REES K. R., ROSS H. F., ROWLAND G. F. The metabolism of isolated rat-kidney nuclei. Biochem J. 1962 Jun;83:523–527. doi: 10.1042/bj0830523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. REES K. R., ROWLAND G. F. The metabolism of isolated rat-liver nuclei. Biochem J. 1961 Jan;78:89–95. doi: 10.1042/bj0780089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. RICHTER D., HULLIN R. P. Isolated nuclei from cells of the cerebral cortex; preparation and enzyme content. Biochem J. 1951 Apr;48(4):406–410. doi: 10.1042/bj0480406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. SCHNEIDER R. M., PETERMANN M. L. Nuclei from normal and leukemic mouse spleen. I. The isolation of nuclei in neutral medium. Cancer Res. 1950 Dec;10(12):751–754. [PubMed] [Google Scholar]
  19. SPORN M. B., WANKO T., DINGMAN W. The isolation of cell nuclei from rat brain. J Cell Biol. 1962 Oct;15:109–120. doi: 10.1083/jcb.15.1.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. STERN H., ALLFREY V., MIRSKY A. E., SAETREN H. Some enzymes of isolated nuclei. J Gen Physiol. 1952 Jan;35(3):559–578. doi: 10.1085/jgp.35.3.559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. STRAUS W. Concentration of acid phosphatase, ribonuclease, desoxyribonuclease, beta-glucuronidase, and cathepsin in droplets isolated from the kidney cells of normal rats. J Biophys Biochem Cytol. 1956 Sep 25;2(5):513–521. doi: 10.1083/jcb.2.5.513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Smyth D. G., Blumenfeld O. O., Konigsberg W. Reactions of N-ethylmaleimide with peptides and amino acids. Biochem J. 1964 Jun;91(3):589–595. doi: 10.1042/bj0910589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. WEISS L. The adhesion of cells. Int Rev Cytol. 1960;9:187–225. doi: 10.1016/s0074-7696(08)62747-3. [DOI] [PubMed] [Google Scholar]

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