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Biochemical Fractionation of Drosophila Cells

  • Melanie Stegman
  • David Robbins
Part of the Methods Inmolecular Biology™ book series (MIMB, volume 397)

Abstract

This chapter describes how to perform basic biochemical fractionations of Drosophila cells, and how to begin to characterize the proteins in the resulting fractions. The protocols include maintenance and transfection of Drosophila cell lines (Section 3.1.), hypotonic lysis (Section 3.2.), and separation of cellular lysates into cytosolic and membrane enriched fractions (Section 3.3.). Cytosolic proteins and those extracted from the membrane enriched fraction can be characterized by size exclusion liquid chromatography (Section 3.4.), while the membrane enriched fraction can be subjected to equilibrium density centrifugation to separate different types of cellular membranes from dense, nonmembranous cellular components (Section 3.5.). The resulting fractions can be used to examine the subcellular localization of a given protein, or the activity of a given protein in various subcellular localizations. When the protein of interest is involved in a signaling pathway, its subcellular localization can provide insight into its mechanism of action in the pathway.

Key Words

Hedgehog subcellular fractionation Drosophila Kinesin signaling methods Costal2 

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Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Melanie Stegman
    • 1
  • David Robbins
    • 2
  1. 1.Department of Microbiology and ImmunologyCornell Weill Medical CollegeNew YorkUSA
  2. 2.Department of Pharmacology and ToxicologyDartmouth Medical SchoolHanoverUSA

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