Biochemical fractionation of Drosophila cells

Melanie Stegman, David J Robbins

Research output: Chapter in Book/Report/Conference proceedingChapter

2 Citations (Scopus)

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
Volume397
DOIs
StatePublished - Sep 13 2007
Externally publishedYes

Publication series

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

Fingerprint

Cell Fractionation
Drosophila
Membranes
Proteins
Centrifugation
Liquid Chromatography
Gel Chromatography
Transfection
Maintenance
Cell Line

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

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

Biochemical fractionation of Drosophila cells. / Stegman, Melanie; Robbins, David J.

Hedgehog Sigaling Protocols. Vol. 397 2007. p. 203-213 (Methods in Molecular Biology; Vol. 397).

Research output: Chapter in Book/Report/Conference proceedingChapter

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