Current Protocols in Cell Biology || Isolation of Nuclei and Nuclear Membranes From Animal Tissues

✍ Scribed by Bonifacino, Juan S.; Dasso, Mary; Harford, Joe B.; Lippincott-Schwartz, Jennifer; Yamada, Kenneth M.

• Book ID: 120935589
• Publisher: Wiley (John Wiley & Sons)
• Year: 2001
• Tongue: English
• Weight: 174 KB
• Volume: 12
• Category: Article
• ISBN-13: 9780471143031
• ISSN: 1934-2500
• DOI: 10.1002/0471143030.cb0310s12

✦ Synopsis

Abstract

The vast majority of methods for the isolation of nuclei and nuclear membranes use soft mammalian tissues (particularly rat liver) as the biological source; however, the protocols described in this unit should be applicable generally to any tissue type, cultured cells, or cells of lower eukaryotes or plants, so long as a suitable homogenization method is available. Generally, a buffered isoosmotic medium containing KCl and MgCl~2~ is used to stabilize the nuclei of mammalian tissues. Some of the media used for cultured animal cells and nonmammalian sources are also described. After homogenization the nuclei are purified using a sucrose barrier of 2.3 M. The use of OptiPrep overcomes the viscosity and osmolarity problems associated with the use of high molarity sucrose, and it permits the use of a high‐speed centrifuge and much shorter centrifugation times. OptiPrep is also used isolate plant cell (i.e., wheat germ) nuclei, because it is able to resolve nuclei from the denser starch granules.