Biochemical fractionation of Drosophila cells

Melanie Stegman, David Robbins

Research output: Chapter in Book/Report/Conference proceedingChapter

2 Scopus citations

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.

Original languageEnglish (US)
Title of host publicationHedgehog Sigaling Protocols
Pages203-213
Number of pages11
DOIs
StatePublished - Sep 13 2007
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume397
ISSN (Print)1064-3745

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Keywords

  • Costal2
  • Drosophila
  • Hedgehog
  • Kinesin
  • Methods
  • Signaling
  • Subcellular fractionation

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Stegman, M., & Robbins, D. (2007). Biochemical fractionation of Drosophila cells. In Hedgehog Sigaling Protocols (pp. 203-213). (Methods in Molecular Biology; Vol. 397). https://doi.org/10.1385/1-59745-516-4:203