Isolation of cajal bodies

Research output: Chapters, Conference Papers, Creative and Literary Works (RGC: 12, 32, 41, 45)12_Chapter in an edited book (Author)peer-review

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Detail(s)

Original languageEnglish
Title of host publicationCell Biology, Four-Volume Set
PublisherElsevier Inc.
Pages115-120
Volume2
ISBN (Print)9780121647308
Publication statusPublished - 2006
Externally publishedYes

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 ~tl of fraction 3S onto a polylysine-coated slide. © 2006 Copyright © 2006 Elsevier Inc. All rights reserved.

Citation Format(s)

Isolation of cajal bodies. / Lam, Yun Wah; Lamond, Angus I.

Cell Biology, Four-Volume Set. Vol. 2 Elsevier Inc., 2006. p. 115-120.

Research output: Chapters, Conference Papers, Creative and Literary Works (RGC: 12, 32, 41, 45)12_Chapter in an edited book (Author)peer-review