Metabolic Labeling and Immunoprecipitation of Drosophila Proteins

Harish Radhakrishna1

1 Georgia Institute of Technology, Atlanta, Georgia
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 7.7
DOI:  10.1002/0471143030.cb0707s12
Online Posting Date:  November, 2001
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Abstract

The genetics of Drosophila is a powerful tool in the analysis of mutants and mutant proteins. Cultures of cells derived from wild‐type or mutant flies can be pulse labeled to biosynthetically label the proteins made by the cells. Immunoprecipitation (and subcellular fractionation) are used to characterize the expression of specific proteins.

     
 
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Table of Contents

  • Basic Protocol 1: Labeling and Immunoprecipitation of Proteins from Drosophila S2M3 Cells
  • Support Protocol 1: Growth and Maintenance of Cultured Drosophila S2M3 Cells
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Labeling and Immunoprecipitation of Proteins from Drosophila S2M3 Cells

  Materials
  • Wild‐type Drosophila S2M3 cells (ATCC CRL 1963) growing in culture in M3 medium (see protocol 2)
  • M3 growth medium (see recipe)
  • M3 labeling medium (see recipe)
  • [35S]L‐Methionine (>800 Ci/mmol) or 35S‐labeled E. coli protein hydrolysate (e.g., TRAN35S‐LABEL, EXPRE35S35S; ICN Biomedicals; >1000 Ci/mmol)
  • Activated charcoal
  • PBS ( appendix 2A), ice‐cold
  • Cell lysis buffer (see recipe)
  • 1:1 (v/v) protein A–Sepharose bead slurry in PBS (store up to 24 hr at 4°C)
  • Specific antibody against protein of interest
  • IP wash buffer: 0.1% (v/v) Triton X‐100 in PBS ( appendix 2A) 1× SDS sample buffer ( appendix 2A)
  • 35‐mm tissue culture dishes
  • Large sealed container (e.g., Tupperware)
  • Cell scrapers
  • End‐over‐end rotator
  • Additional reagents and equipment for culturing Drosophila cells (see protocol 2)
CAUTION: When working with radioactive materials, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following guidelines provided by the local radiation safety officer (also see unit 7.1 and appendix 1D).

Support Protocol 1: Growth and Maintenance of Cultured Drosophila S2M3 Cells

  Materials
  • Drosophila S2M3 cells (ATCC CRL 1963)
  • M3 growth medium (see recipe)
  • 25‐cm2 (T‐25) tissue culture flasks with plug‐seal caps
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Figures

Videos

Literature Cited

Literature Cited
   Bunch, T., Grinblat, Y., and Goldstein, L.S. 1988. Characterization and use of the Drosophila metallothionein promoter in cultured Drosophila melanogaster cells. Nucl. Acids Res. 16:1043‐1061.
   Buzin, C.H. and Petersen, N.S. 1982. A comparison of the multiple heat‐shock proteins in cell lines and larval salivary glands by two‐dimensional gel electrophoresis. J. Mol. Biol. 158:181‐201.
   Chen, C. and Okayama, H. 1987. High‐efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7:2745‐2752.
   Cherbas, L., Moss, R., and Cherbas, P. 1994. Transformation techniques for Drosophila cell lines. Methods Cell Biol. 44:161‐179.
   Echalier, G. 1997. Drosophila Cells in Culture. Academic Press, San Diego.
   Echalier, G. and Ohanessian, A. 1970. In vitro culture of Drosophila melanogaster embryonic cells. In Vitro 6:162‐172.
   Jaynes, J.B. and O'Farrell, P.H. 1988. Activation and repression of transcription by homeodomain‐containing proteins that bind a common site. Nature 336:744‐749.
   Schneider, I. 1972. Cell lines derived from late embryonic stages of Drosophila melanogaster. J. Embryol. Exp. Morphol. 27:353‐365.
   Zak, N.B. and Shilo, B.Z. 1990. Biochemical properties of the Drosophila EGF receptor homolog (DER) protein. Oncogene 5:1589‐1593.
Key Reference
   Echalier, G. 1997. See above.
  An excellent and comprehensive description of Drosophila cell culture protocols that includes methods for generating both primary and continuous cell lines, media formulations, and experimental uses of Drosophila cells.
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