Abstract
The nucleolus is involved in a number of cellular processes of importance to cell physiology and pathology, including cell stress responses and malignancies. Studies of macromolecular composition of the nucleolus depend critically on the efficient extraction and accurate quantification of all macromolecular components (e.g., DNA, RNA, and protein). We have developed a TRIzol-based method that efficiently and simultaneously isolates these three macromolecular constituents from the same sample of purified nucleoli. The recovered and solubilized protein can be accurately quantified by the bicinchoninic acid assay and assessed by polyacrylamide gel electrophoresis or by mass spectrometry. We have successfully applied this approach to extract and quantify the responses of all three macromolecular components in nucleoli after drug treatments of HeLa cells, and conducted RNA-Seq analysis of the nucleolar RNA.
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Acknowledgements
The original work leading to the development of this protocol has been supported by NIH P30 CA006973 and by Johns Hopkins University start-up funds.
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Bai, B., Laiho, M. (2015). Sequential Recovery of Macromolecular Components of the Nucleolus. In: Hancock, R. (eds) The Nucleus. Methods in Molecular Biology, vol 1228. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1680-1_5
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DOI: https://doi.org/10.1007/978-1-4939-1680-1_5
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1679-5
Online ISBN: 978-1-4939-1680-1
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