Large-scale isolation of Cajal bodies from HeLa cells.
The Cajal body (CB) is a conserved, dynamic nuclear structure that is implicated in various cellular processes, such as the maturation of splicing small nuclear ribonucleoproteins 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 electron microscopy analysis, are also similar to those 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 characterization of their molecular composition, structure, and properties.
Lam
Yun Wah
YW
Wellcome Trust Biocentre, MSI/WTB Complex, University of Dundee, Dundee DD1 4HN, Scotland, United Kingdom.
Lyon
Carol E
CE
Lamond
Angus I
AI
D000818:Animals; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism*; D020541:Coiled Bodies / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D008274:Magnesium / Q000378:metabolism; D009687:Nuclear Proteins / Q000302:isolation & purification* / Q000378:metabolism; D013010:Sonication
D009687:Nuclear Proteins; C068921:p80-coilin; D008274:Magnesium