Isolation of nuclei and nuclear membranes from animal tissues

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 MgCl2 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.