Abstract
The integrity of a subcellular proteome such as the nucleus, is largely dependent on purification of the isolated compartment away from other cellular contaminants. The separation of high-purity nuclei from plants is a difficult task. However, successful purification has been achieved through a series of fractionation processes. Initially, centrifugation in a 2.0 M sucrose density gradient (1) or a percoll density gradient (2) was used to isolate nuclei from cultured rice suspension cells. A modified version of the sucrose gradient method described in Morre and Anderson (1) has proved to be more rapid and efficient for the isolation of nuclei from cultured rice suspension cells. The nuclei are uniform spheres with an average diameter of approx 20 µm. The nuclear proteins were prepared from the purified nuclei using lysis buffer (3) or SDS sample buffer (4). The purity of the isolated nuclear fraction was evaluated by Western blot analysis using antihistone H1 antibody, a specific antibody for nuclear proteins. Histone H1 was found in the nuclear fraction, but not in the supernatant fraction, suggesting that the preparation is enriched in nuclear proteins.
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References
Morre, D. J. and Andersson, B. (1994) Isolation of all major organelles and membranous cell components from a single homogenate of green leaves. Methods Enzymol. 228, 412–419.
Folta, K. M. and Kaufman, L. S. (2000) Preparation of transcriptionally active nuclei from etiolated Arabidopsis thaliana. Plant Cell Rep. 19, 504–510.
O’Farrell, P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.
Darnell, J., Lodish, H., and Baltimore, D. (1990) in Molecular Cell Biology, Scientific American Books, New York.
Khan, M. and Komatsu, S. (2004) Rice proteomics: recent developments and analysis of nuclear proteins. Phytochemistry 65, 1671–1681.
Bae, M. S., Cho, E. J., Choi, E.-Y., and Park, O. K. (2003) Analysis of the Arabidopsis nuclear proteome and its response to cold stress. Plant J. 36, 652–663.
Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol. Plant 15, 473–497.
Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.
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© 2007 Humana Press Inc., Totowa, NJ
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Komatsu, S. (2007). Extraction of Nuclear Proteins. In: Thiellement, H., Zivy, M., Damerval, C., Méchin, V. (eds) Plant Proteomics. Methods in Molecular Biology, vol 355. Humana Press. https://doi.org/10.1385/1-59745-227-0:73
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DOI: https://doi.org/10.1385/1-59745-227-0:73
Publisher Name: Humana Press
Print ISBN: 978-1-58829-635-1
Online ISBN: 978-1-59745-227-4
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