Isolation of cajal bodies

Yun Wah Lam (Lead / Corresponding author), Angus I. Lamond

    Research output: Chapter in Book/Report/Conference proceedingChapter

    Abstract

    This chapter describes an effective procedure for the large-scale isolation of Cajal bodies (CBs) from mammalian somatic cell nuclei. Density separation is carried out using Percoll, a silica sol coated with polyvinylpyrolidone, which generates a density gradient on ultracentrifugation. It results in the enrichment of particles containing known CB factors that are comparable in size, morphology, and composition to CBs detected in situ. Prepare the starting material of HeLa nuclei. Divide the diluted nuclei into 2 × 15 ml portions. Overlay each portion onto 15 ml of the S2 solution in a 50-ml Falcon tube. Make sure the interface of the two layers is sharp. Examine the sonicated nuclei under a phasecontrast microscope. The majority of nuclei should have been lyzed, while nucleoli should be clearly visible. A sonicator probe that has been used repeatedly develops pits on its end. The sonication efficiency gradually decreases as time goes on. Therefore, the sonication time recommended here can only be used as a guideline. To immunolabel the isolated CBs, spot about 5 μl of fraction 3S onto a polylysine-coated slide. 

    Original languageEnglish
    Title of host publicationCell Biology, Four-Volume Set
    EditorsJulio E. Celis
    PublisherElsevier Inc.
    Pages115-120
    Number of pages6
    Volume2
    Edition3rd
    ISBN (Print)9780121647308
    DOIs
    Publication statusPublished - 1 Dec 2006

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