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Pauly_Fishing_Down_Marine_Food_Webs

Pauly_Fishing_Down_Marine_Food_Webs - Fishing Down Marine...

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DOI: 10.1126/science.279.5352.860 , 860 (1998); 279 Science et al. Daniel Pauly, Fishing Down Marine Food Webs www.sciencemag.org (this information is current as of November 21, 2007 ): The following resources related to this article are available online at http://www.sciencemag.org/cgi/content/full/279/5352/860 version of this article at: including high-resolution figures, can be found in the online Updated information and services, found at: can be related to this article A list of selected additional articles on the Science Web sites http://www.sciencemag.org/cgi/content/full/279/5352/860#related-content http://www.sciencemag.org/cgi/content/full/279/5352/860#otherarticles , 1 of which can be accessed for free: cites 5 articles This article 591 article(s) on the ISI Web of Science. cited by This article has been http://www.sciencemag.org/cgi/content/full/279/5352/860#otherarticles 49 articles hosted by HighWire Press; see: cited by This article has been http://www.sciencemag.org/cgi/collection/ecology Ecology : subject collections This article appears in the following http://www.sciencemag.org/about/permissions.dtl in whole or in part can be found at: this article permission to reproduce of this article or about obtaining reprints Information about obtaining registered trademark of AAAS. is a Science 1998 by the American Association for the Advancement of Science; all rights reserved. The title Copyright American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the Science on November 21, 2007 www.sciencemag.org Downloaded from
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PRP24 fragment or a 1.4-kb Apa I–Sal I PRP24-Pya fragment ( 13 ). Both constructs overexpressed Prp24p when transformed into PRY98 as deter- mined by Western blotting with anti-Prp24 polyclonal antibodies. 19. Three liters of each strain, PRY115 (PRY98 with pG1-PRP24 as sole PRP24 gene) and PRY116 (PRY98 with pG1-PRP24-Pya as sole PRP24 gene), were harvested in late logarithmic phase. Whole cell extract was prepared ( 15 ), and 150 mg of each was subjected to 30 to 55% ammonium sulfate precipitation. The precipitates were resus- pended in 40 ml of AGK 200 [10 mM Hepes (pH 7.9), 200 mM KCl, 1.5 mM MgCl 2 , 10% glycerol, 0.2 mM EDTA, 0.1% Triton X-100, 0.5 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzami- dine, pepstatin A (1 g/ml), leupeptin (1 g/ml)] and incubated with 150 l of protein G–Sepharose (washed in AGK 200) on a nutator at 4°C for 30 min. ( This step removed proteins that bound non- specifically to the resin.) The supernatants were added to 1 ml of protein G–Sepharose coupled to anti-polyoma antibodies (beads: AGK 200 1:1) as in ( 32 ). After 1.5 hours nutating at 4°C, the supernatants were removed, and the beads were washed 5 20 ml of AGK 700 (AGK buffer with 700 mM KCl), 1 15 ml of AGK 200, and 1 15 ml of AGK 50 (AGK buffer with 50 mM KCl). The washed beads were eluted twice by nutating at 23°C for 10 min with 2 400 l (1 mg/ml) of peptide EYMPME (Glu-Tyr-Met-Pro-Met-Glu) ( 32 ) in AGK 50. Each eluate was dialyzed against 2 2 liters of buffer D at 4°C for 3.5 hours. The dialyzates were microcentrifuged 10 min, and the superna-
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