Affinity purification of FLAG - tagged protein complexes : A cautionary tale
نویسندگان
چکیده
Through two simple changes in the composition of the fixative for freeze-substitution of cryoimmobilized specimens, we are able to ameliorate the reproducible visibility of the plasma membrane in D. melanogaster embryos and larvae. The ultrastructural analysis of membrane-defective phenotypes of embryos fixed by our modified method should allow us a refined view on the plasma membrane and give us new insight in the function of the respective factors in particular and in membrane biology in general. Acknowledgments: We are grateful to the technical assistance of Frauke Meyer, Ursula Müller, and Brigitte Sailer. This work was supported by the German Research Foundation (DFG, MO1714/2-1). References: Buser, C., and P. Walther 2008, J. Microsc. 230: 268-277; McDonald, K., and M.K. Morphew 1993, Microsc. Res. Tech. 24: 465-473; McDonald, K., and T. Muller-Reichert 2008, J. Microsc. 230: 252; McDonald, K.L., 2009, J. Microsc. 235: 273-281; McDonald, K.L., M. Morphew, P. Verkade, and T. Muller-Reichert 2007, Methods Mol. Biol. 369: 143-173; Moussian, B., 2010, Insect Biochem. Mol. Biol. 40: 363-375; Moussian, B., H. Schwarz, S. Bartoszewski, and C. Nusslein-Volhard 2005a, J. Morphol. 264: 117-130; Moussian, B., C. Seifarth, U. Muller, J. Berger, and H. Schwarz 2006a, Arthropod Struct. Dev. 35: 137-152; Moussian, B., J. Soding, H. Schwarz, and C. Nusslein-Volhard 2005b, Dev. Dyn. 233: 1056-1063; Moussian, B., E. Tang, A. Tonning, S. Helms, H. Schwarz, C. Nusslein-Volhard, and A.E. Uv 2006b, Development 133: 163171; Moussian, B., J. Veerkamp, U. Muller, and H. Schwarz 2007, Matrix Biol. 26: 337-347; Steinbrecht, R.A., 1980, Tissue Cell, 12: 73-100; Studer, D., M. Michel, and M. Muller 1989, Scanning Microsc. Suppl. 3: 253-268, discussion 268-259; Tonning, A., S. Helms, H. Schwarz, A.E. Uv, and B. Moussian 2006, Development 133: 331-341; Uv, A., and B. Moussian 2010, Eur. J. Cell Biol. 89: 208-211; Walther, P., and A. Ziegler 2002, J. Microsc. 208: 3-10; Zhang, S., and E.H. Chen 2008, Methods Mol. Biol. 475: 275-297.
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