Immunoisolation of centrosomes from Drosophila melanogaster.
Classical protocols for the isolation of centrosomes from higher eukaryotic cells are based on enrichment of cell organelles by density gradient centrifugation. Various successful protocols have been described that isolate centrosomes from mammalian tissue culture cells, tissue, clam oocytes, Drosophila, and yeast, to mention only some of the more frequently used sources. The material produced is subsequently used in various assays. These include functional tests such as the microtubule nucleation assay, electron microscopic study of centrosome morphology, and antigen localization; the organelles may also be used for the generation of antibodies. Furthermore, centrosomal preparations have been used for the characterization of their protein composition. The method described here focuses on the isolation of centrosomes from the syncytial stages of the early Drosophila embryo. This is a particularly attractive system because these organelles are not bounded by cellular membranes. Moreover, the abundance of pericentriolar material of these centrosomes produces excellent total protein yields.
Lehmann
Verena
V
Max Planck Institute for Molecular Genetics, Berlin, Germany.
Müller
Hannah
H
Lange
Bodo M H
BM
D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002499:Centrifugation, Density Gradient / Q000379:methods*; D018385:Centrosome / Q000276:immunology / Q000648:ultrastructure*; D004331:Drosophila melanogaster; D004625:Embryo, Nonmammalian / Q000166:cytology*; D005455:Fluorescent Antibody Technique