Karyoskeletal protein-enriched fraction Drosophila

Preparation of Karyoskeletal Protein-Enriched Fractions from Drosophila melanogaster Cells and Tissues... 

Unless otherwise noted, all procedures should be performed at 4°C or on ice. Nuclear purification is the first step in preparation of karyoskeletal protein-enriched fractions from Drosophila cells and tissues. Two techniques have been used extensively in our laboratory. The first, as detailed previously in Fisher et al. (1982), is particularly useful for preparation of undegraded karyoskeletal proteins from embryos without regard for their biological activity. 

Fig. 2 Transmission electron micrograph of karyoskeletal protein-enriched fraction prepared from Drosophila embryos. The main panel shows a nucleuslike structure, bounded by a peripheral lamina (L) Inset Higher magnification showing tangential section through the periphery of karyoskeletal protein structure as shown in the main panel. Nuclear pore complex remnants can be readily appreciated as small ringlike structures.

Fig. 3 SDS-gradient PAGE and Coomassie blue staining. Lane 1, Final karyoskeletal protein-enriched fraction after nuclease digestion at 37°C lane 2,1 Af NaCl extract after nuclease digestion at 23°C. Migration positions of various Drosophila marker proteins are indicated to the right of the figure.

Figure 2 is a transmission electron micrograph of a nuclease-treated, Triton X-lOO-extracted, twice NaCl-extracted karyoskeletal protein-enriched fraction derived from Drosophila embryos. Nuclease treatment was at 37°C. Identifiable karyoskeletal elements are labeled. The SDS-PAGE profiles of the final karyoskeletal protein-enriched pellet fraction generated after subfractionation of nuclei treated with nucleases at 37°C are shown in Fig. 3 (lane 1) as well as the first 1 M NaCl extract generated after subfractionation of nuclei treated with nucleases at 23°C (also highly enriched for Drosophila karyoskeletal proteins but in soluble form) (lane 2). 

Fisher, P. A., Lin, L., McConneU, M., Greenleaf, A., Lee, J.-M., and Smith, D. E. (1989). Heat shock-induced appearance of RNA polymerase II in karyoskeletal protein-enriched (nuclear matrix ) fractions correlates with transcriptional shutdown in Drosophila melanogaster. J. Biol. Chem. 264, 3464-3469,... 

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