Skip to main content

Purification of Nuclei and Preparation of Nuclear Envelopes from Skeletal Muscle

  • Protocol
The Nucleus

Part of the book series: Methods in Molecular Biology ((MIMB,volume 463))

Abstract

The nuclear envelope is a complex membrane-protein system that is notoriously difficult to purify because it has many connections to both nuclear and cytoplasmic components. This difficulty is compounded by the fact that the nature of these connections vary in different cell types, and so methods must be significantly adapted according to the cell type from which nuclear envelopes are being purified. Here we present a detailed method for purification of nuclear envelopes from one of the most intransigent tissues: skeletal muscle. We further note in the procedure how this method differs from that for other tissues. Identification of nuclear envelope-specific proteins is principally encumbered by endoplasmic reticulum contamination; therefore, we also present a method to purify sarcoplasmic reticulum from muscle.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Hetzer, M. W., Walther, T. C., and Mattaj, I. W. (2005) Pushing the envelope: structure, function, and dynamics of the nuclear periphery. Annu. Rev. Cell Dev. Biol. 21, 347–380.

    Article  PubMed  CAS  Google Scholar 

  2. Mattout, A., Dechat, T., Adam, S. A., Goldman, R. D., and Gruenbaum, Y. (2006) Nuclear lamins, diseases and aging. Curr. Opin. Cell Biol. 18, 335–341.

    Article  PubMed  CAS  Google Scholar 

  3. Schirmer, E. C. and Gerace, L. (2005) The nuclear membrane proteome: extending the envelope. Trends Biochem. Sci. 30, 551–558.

    Article  PubMed  CAS  Google Scholar 

  4. Roux, K. J. and Burke, B. (2007) Nuclear envelope defects in muscular dystrophy. Biochim. Biophys. Acta. 1772, 118–127.

    Article  PubMed  CAS  Google Scholar 

  5. Wilkie, G. S. and Schirmer, E. C. (2006) Guilt by association: the nuclear envelope proteome and disease. Mol. Cell Proteomics 5, 1865–1875.

    Article  PubMed  CAS  Google Scholar 

  6. Starr, D. A. and Han, M. (2002) Role of ANC-1 in tethering nuclei to the actin cytoskeleton. Science 298, 406–409.

    Article  PubMed  CAS  Google Scholar 

  7. Crisp, M., Liu, Q., Roux, K., Rattner, J. B., Shanahan, C., Burke, B., Stahl, P. D., and Hodzic, D. (2006) Coupling of the nucleus and cytoplasm: role of the LINC complex. J. Cell Biol. 172, 41–53

    Article  PubMed  CAS  Google Scholar 

  8. Houben, F., Ramaekers, F. C., Snoeckx, L. H., and Broers, J. L. (2007) Role of nuclear lam-ina-cytoskeleton interactions in the maintenance of cellular strength. Biochim. Biophys. Acta 1773, 675–686.

    Article  PubMed  CAS  Google Scholar 

  9. Mattout-Drubezki, A. and Gruenbaum, Y. (2003) Dynamic interactions of nuclear lamina proteins with chromatin and transcriptional machinery. Cell Mol. Life Sci. 60, 2053–2063.

    Article  PubMed  CAS  Google Scholar 

  10. Stuurman, N., Heins, S., and Aebi, U. (1998) Nuclear lamins: their structure, assembly, and interactions. J. Struct. Biol. 122, 42–66.

    Article  PubMed  CAS  Google Scholar 

  11. Blobel, G. and Potter, V. R. (1966) Nuclei from rat liver: isolation method that combines purity with high yield. Science 154, 1662–1665.

    Article  PubMed  CAS  Google Scholar 

  12. Aaronson, R. P. and Blobel, G. (1975) Isolation of nuclear pore complexes in association with a lamina. Proc. Natl. Acad. Sci. USA 72, 1007–1011.

    Article  PubMed  CAS  Google Scholar 

  13. Dwyer, N. and Blobel, G. (1976) A modified procedure for the isolation of a pore complex-lamina fraction from rat liver nuclei. J. Cell Biol. 70, 581–591.

    Article  PubMed  CAS  Google Scholar 

  14. Gerace, L., Ottaviano, Y., and Kondor-Koch, C. (1982) Identification of a major polypeptide of the nuclear pore complex. J. Cell Biol. 95, 826–837.

    Article  PubMed  CAS  Google Scholar 

  15. Florens, L., Korfali, N., and Schirmer, E. C. (2008) Subcellular fractionation and proteomics of nuclear envelopes, in Methods in Molecular Biology. Humana, Totowa, NJ, 432, 117–137.

    Google Scholar 

  16. Korfali, N., Fairley, E. A., Swanson, S. K., Florens, L., and Schirmer, E. C. (2009) Use of sequential chemical extractions to purify nuclear membrane proteins for proteomics identification, in Methods in Molecular Biology. Humana, Totowa, NJ, pp (in press).

    Google Scholar 

  17. Kuehl, L. (1977) Isolation of skeletal muscle nuclei. Methods Cell Biol. 15, 79–88.

    Article  PubMed  CAS  Google Scholar 

  18. Kuehl, L. (1975) Isolation of skeletal muscle nuclei. Exp. Cell Res. 91, 441–448.

    Article  PubMed  CAS  Google Scholar 

  19. Hahn, C. G. and Covault, J. (1990) Isolation of transcriptionally active nuclei from striated muscle using Percoll density gradients. Anal. Biochem. 190, 193–197.

    Article  PubMed  CAS  Google Scholar 

  20. Liew, C. C., Jackowski, G., Ma, T., and Sole, M. J. (1983) Nonenzymatic separation of myo-cardial cell nuclei from whole heart tissue. Am. J. Physiol. 244, C3–10.

    PubMed  CAS  Google Scholar 

  21. Dreger, M., Bengtsson, L., Schoneberg, T., Otto, H., and Hucho, F. (2001) Nuclear envelope proteomics: novel integral membrane proteins of the inner nuclear membrane. Proc. Natl. Acad. Sci. USA 98, 11943–11948.

    Article  PubMed  CAS  Google Scholar 

  22. Cronshaw, J., Krutchinsky, A., Zhang, W., Chait, B., and Matunis, M. (2002) Proteomic analysis of the mammalian nuclear pore complex. J. Cell Biol. 158, 915–927.

    Article  PubMed  CAS  Google Scholar 

  23. Schirmer, E. C., Florens, L., Guan, T., Yates, J. R., and Gerace, L. (2003) Nuclear membrane proteins with potential disease links found by subtractive proteomics. Science 301, 1380–1382.

    Article  PubMed  CAS  Google Scholar 

  24. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York.

    Google Scholar 

  25. Aaronson, R. P. and Blobel, G. (1974) On the attachment of the nuclear pore complex. J. Cell Biol. 62, 746–754.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2008 Humana Press

About this protocol

Cite this protocol

Wilkie, G.S., Schirmer, E.C. (2008). Purification of Nuclei and Preparation of Nuclear Envelopes from Skeletal Muscle. In: Hancock, R. (eds) The Nucleus. Methods in Molecular Biology, vol 463. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-406-3_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-406-3_2

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-58829-977-2

  • Online ISBN: 978-1-59745-406-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics