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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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.
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.
Schirmer, E. C. and Gerace, L. (2005) The nuclear membrane proteome: extending the envelope. Trends Biochem. Sci. 30, 551–558.
Roux, K. J. and Burke, B. (2007) Nuclear envelope defects in muscular dystrophy. Biochim. Biophys. Acta. 1772, 118–127.
Wilkie, G. S. and Schirmer, E. C. (2006) Guilt by association: the nuclear envelope proteome and disease. Mol. Cell Proteomics 5, 1865–1875.
Starr, D. A. and Han, M. (2002) Role of ANC-1 in tethering nuclei to the actin cytoskeleton. Science 298, 406–409.
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
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.
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.
Stuurman, N., Heins, S., and Aebi, U. (1998) Nuclear lamins: their structure, assembly, and interactions. J. Struct. Biol. 122, 42–66.
Blobel, G. and Potter, V. R. (1966) Nuclei from rat liver: isolation method that combines purity with high yield. Science 154, 1662–1665.
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.
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.
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.
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.
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).
Kuehl, L. (1977) Isolation of skeletal muscle nuclei. Methods Cell Biol. 15, 79–88.
Kuehl, L. (1975) Isolation of skeletal muscle nuclei. Exp. Cell Res. 91, 441–448.
Hahn, C. G. and Covault, J. (1990) Isolation of transcriptionally active nuclei from striated muscle using Percoll density gradients. Anal. Biochem. 190, 193–197.
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.
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.
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.
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.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York.
Aaronson, R. P. and Blobel, G. (1974) On the attachment of the nuclear pore complex. J. Cell Biol. 62, 746–754.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights 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