Immunoisolation of Centrosomes from Drosophila melanogaster

Abstract

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.

Evidence that a positive feedback loop drives centrosome maturation in fly embryos

“…Whole centrosomes were isolated from extracts of early Drosophila embryos (0–4 hr) using a modified version of a centrosome isolation protocol (Lehmann et al, 2006). Embryo extract containing 50% sucrose was layered on top of a sucrose cushion comprising 55% and 70% sucrose.…”

Functional Analysis of Centrosomal Kinase Substrates in Drosophila melanogaster Reveals a New Function of the Nuclear Envelope Component Otefin in Cell Cycle Progression

“…In parallel, immunofluorescence microscopy of centrosomes from all isolation steps using anti-␥-Tub antibody was performed as described before (36).…”

Cloning and characterization of a sialic acid binding lectins (SABL) from Manila clam Venerupis philippinarum

“…Clams, without the benefit of an adaptive immune system, possess an immensely strong innate immune response to counteract the continuous challenge of infection in marine environment [1]. Lectin, the pattern recognition protein (PRP) recognizing and binding to terminal sugars on glycoproteins and glycolipids, is one of the major components in these host-defence systems [2].…”

“…However, little information is available about the molecular features and immune response against pathogen infection in commercially cultured clam Venerupis philippinarum. The main objectives of this study are: (1) to clone the full-length cDNA of SABL from V. philippinarum (VpSABL), (2) to examine the tissue expression profile of VpSABL in healthy clam, (3) to investigate the expression profile of VpSABL after being infected by Vibrio pathogen.…”