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MBC in Press, published online ahead of print April 24, 2002
Mol. Biol. Cell 10.1091/mbc.02-03-0034

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Submitted on December 4, 2001
Revised on February 20, 2002
Accepted on April 5, 2002

Large-scale isolation of Cajal bodies from HeLa cells

Yun Wah Lam1, Carol E. Lyon1, and Angus I. Lamond1*

1 Wellcome Trust Biocentre, MSI/WTB Complex, University of Dundee, Dundee DD1 4HN, Scotland, United Kingdom

* Corresponding author. E-mail address: a.i.lamond{at}dundee.ac.uk.

The Cajal body (CB) is a conserved, dynamic nuclear structure that is implicated in various cellular processes such as the maturation of splicing snRNPs and the assembly of transcription complexes. Here we report the first procedure for the large-scale purification of CBs from HeLa cell nuclei, resulting in an approximately 750-fold enrichment of the CB marker protein p80-coilin. Immunofluorescence, immunoblotting and mass spectrometric analyses showed that the composition of the isolated CBs was similar to that of CBs in situ. The morphology and structure of the isolated CBs, as judged by transmission and scanning EM analysis, are also similar to that of CBs in situ. This protocol demonstrates the feasibility of isolating intact distinct classes of subnuclear bodies from cultured cells in sufficient yield and purity to allow detailed characterisation of their molecular composition, structure and properties.




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