Extraction of nuclear proteins.
The integrity of a subcellular proteome such as the nucleus, is largely dependent on purification of the isolated compartment away from other cellular contaminants. The separation of high-purity nuclei from plants is a difficult task. However, successful purification has been achieved through a series of fractionation processes. Initially, centrifugation in a 2.0 M sucrose density gradient (1) or a percoll density gradient (2) was used to isolate nuclei from cultured rice suspension cells. A modified version of the sucrose gradient method described in Morre and Anderson (1) has proved to be more rapid and efficient for the isolation of nuclei from cultured rice suspension cells. The nuclei are uniform spheres with an average diameter of approx 20 microm. The nuclear proteins were prepared from the purified nuclei using lysis buffer (3) or SDS sample buffer (4). The purity of the isolated nuclear fraction was evaluated by Western blot analysis using antihistone H1 antibody, a specific antibody for nuclear proteins. Histone H1 was found in the nuclear fraction, but not in the supernatant fraction, suggesting that the preparation is enriched in nuclear proteins.
Komatsu
Setsuko
S
Laboratory of Gene Regulation, Department of Molecular Genetics, National Institute of Agrobiological Sciences, Tsukuba, Japan.
D015153:Blotting, Western; D002021:Buffers; D002458:Cell Fractionation / Q000379:methods; D002467:Cell Nucleus / Q000737:chemistry; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional; D006657:Histones / Q000032:analysis; D009687:Nuclear Proteins / Q000302:isolation & purification*; D012275:Oryza / Q000737:chemistry; D010940:Plant Proteins / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*
D002021:Buffers; D006657:Histones; D009687:Nuclear Proteins; D010940:Plant Proteins