Drosophila extract preparation

To purify lamins. Drosophila extracts prepared as described above should be supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF). Lamins from these extracts should be batch adsorbed to antilamin/protein A/sepharose by incubation overnight at 4°C. The column should then be washed extensively with equilibration buffer and purified lamins recovered from the antilamin affinity column with elution buffer. After elution, the solution containing the lamins should be immediately neutralized with Na2HP04 added to a final concentration of 50 mM. Immunoaffinity-purified lamins should be aliquoted, frozen by immersion in liquid nitrogen, and stored at -70°C until use. 

We have been able to purify heat shock activator protein, a 110 kD polypeptide, to over 95% homogeneity from a 0.35 M NaCl nuclear extract prepared from heat shocked Drosophila tissue culture cells (3). The extract was chromatographed on heparin-Sepharose, twice sequentially on specific DNA-Sepharose (4 ml and 0.6 ml bed volumes), and on FPLC Mono S (a Pharmacia ion exchange matrix). A typical purification requires 1.5 x 10 cells from which several micrograms of homogenous heat shock activator protein are obtained. The yield at the affinity step is > 50%. Typical overall yields range between 10% and 20%. The... 

To prepare extracts, between 100 and 500 virgin Drosophila females, 7-10 days old, should be anesthetized by chilling and placed in a plastic petri dish. 

Extracts from Drosophila melanogaster (Oregon R, P2 strain) embryos can be prepared in the following way (Berrios and Avilion, 1990 Kawasaki et ai,... 

The preparation of extracts from preblastoderm Drosophila embryos and their use for assembling chromatin has been described in detail by Becker et al. (1994). In this section I briefly outline these procedures as well as the in vitro conditions under which nucleosome remodeling occurs. 

In addition, we present methods for the preparation of HeLa cell extracts that can support import in vitro. In general, we find for both proteins and snRNPs that the Xenopus egg extract is the most efficient in terms of the speed of nuclear import and the final level of intranuclear accumulation. The Xenopus oocyte extract, HeLa cell extract, and reticulocyte lysates are generally less efficient in our hands. We have not been able to reconstitute nuclear import using extracts from Drosophila early embryos (C. Dingwall, unpublished). 

Preparation of Nuclear Extracts from Drosophila Embryos and In Vitro Transcription Analysis ... 

The production of a high-quahty Drosophila embryonic cytoplasmic extract for use in protein purification or biochemistry is relatively easy, providing population cages are available that produce at least 5 g of embryos during a 3-hour period (for methods to maintain population cages, see Sisson, this volume). Smaller quantities of embryos can be used to produce extracts that are useftil for in vitro biochemical assays (see, e.g., Moritz et al. 1998), and thus it should be possible to make extracts from mutant stocks that could then be tested in vitro. Protocol 33.1 describes the collection and dechorionation of embryos for making cytoplasmic extracts. It is fairly easy to prepare even very concentrated cytoplasmic extracts from Drosophila embryos, with protein concentrations of 50-75 mg/ml (see Protocol 33.2). 

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