Preparation of a Nuclear Matrix-Pore Complex-Lamina Fraction from Embryos of Drosophila melanoǵaster

This chapter describes the methodology used for preparation of a nuclear matrix-pore complex-lamina fraction from embryos of Drosophila melanogaster. The procedure outlined in the chapter for the purification of nuclei from D.melanogaster embryos is simple, rapid, and efficient; the nuclei obtained from this procedure are free of detectable cytoplasmic contaminants as assessed both by phase contrast light microscopy and transmission electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) reveals a polypeptide composition of the purified nuclei, completely distinct from that of the postnuclear supernatant. Immunochemical analyses and direct examination of polypeptide patterns at each step of the fractionation procedure demonstrate that, in the presence of all three protease inhibitors contained in buffer A, there is little or no proteolysis taking place during the fractionation of Drosophila embryos. Thus, maintenance of chromatin structure through the nuclease digestion step may prove to be crucial to the efficient preparation of a stable nuclear matrix.