A New Rapid Method for Isolating Nucleoli

Zhou Fang Li, Yun Wah Lam

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

20 Citations (Scopus)

Abstract

The nucleolus was one of the first subcellular organelles to be isolated from the cell. The advent of modern proteomic techniques has resulted in the identification of thousands of proteins in this organelle, and live cell imaging technology has allowed the study of the dynamics of these proteins. However, the limitations of current nucleolar isolation methods hinder the further exploration of this structure. In particular, these methods require the use of a large number of cells and tedious procedures. In this chapter we describe a new and improved nucleolar isolation method for cultured adherent cells. In this method cells are snapfrozen before direct sonication and centrifugation onto a sucrose cushion. The nucleoli can be obtained within a time as short as 20 min, and the high yield allows the use of less starting material. As a result, this method can capture rapid biochemical changes in nucleoli by freezing the cells at a precise time, hence faithfully refl ecting the protein composition of nucleoli at the specified time point. This protocol will be useful for proteomic studies of dynamic events in the nucleolus and for better understanding of the biology of mammalian cells.
Original languageEnglish
Title of host publicationThe Nucleus
EditorsRonald Hancock
PublisherHumana Press Inc.
Pages35-42
Number of pages8
VolumeMIMB 1228
Edition2nd
ISBN (Electronic)9781493916801
ISBN (Print)9781493916795, 9781493948734
DOIs
Publication statusPublished - 14 Oct 2014
Externally publishedYes

Publication series

NameMethods in Molecular Biology
PublisherHumana Press
VolumeMIMB 1228
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Cite this