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 ?l of fraction 3S onto a polylysine-coated slide.
|Title of host publication||Cell biology|
|Subtitle of host publication||A laboratory handbook|
|Editors||Julio E. Celis|
|Number of pages||6|
|Publication status||Published - 16 Nov 2005|