Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation

Bartholomew M. Sefton1

1 The Salk Institute, San Diego, California
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 14.4
DOI:  10.1002/0471143030.cb1404s03
Online Posting Date:  May, 2001
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Abstract

Signal transduction pathways may involve protein phosphorylation at one or more residues. Detection of phosphorylation involves labeling with radioactive inorganic phosphate and subsequent immunoprecipitation with appropriate antibodies. This unit describes labeling conditions for adherent and nonadherent cells and preparation of the lysates from these cells for immunoprecipitation. The labeling method is appropriate for labeling other phosphorylated component of cells, but alternate methods are necessary for their detection.

     
 
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Table of Contents

  • Basic Protocol 1: Labeling Cultured Cells with 32Pi and Lysis Using Mild Detergent
  • Alternate Protocol 1: Lysis of Cells by Boiling in SDS
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Labeling Cultured Cells with 32Pi and Lysis Using Mild Detergent

  Materials
  • Cell culture to be labeled
  • Labeling medium: phosphate‐free tissue culture medium (e.g., DMEM, appendix 2A) supplemented with the usual concentration of serum or serum dialyzed against phosphate‐free saline, 37°C
  • 500 mCi/ml to 1 Ci/ml H 332PO 4 in HCl (carrier free ICN)
  • Tris‐buffered (TBS; see recipe) or Dulbecco's phosphate‐buffered saline (DPBS; appendix 2A), cold
  • Mild lysis buffer or RIPA lysis buffer (see reciperecipes)
  • 1‐in.‐thick Plexiglas shield ( appendix 1D)
  • Plugged, aerosol‐resistant pipet tips
  • Plexiglas box ( appendix 1D), warmed to 37°C
  • Screw‐cap microcentrifuge tubes
  • Plugged disposable pipet or disposable one‐piece transfer pipet
  • Rubber policeman
  • Sorvall refrigerated centrifuge with SM 24 rotor and rubber adaptors, refrigerated microcentrifuge, or equivalent, 4°C
  • Plexiglas sheet (10 × 10 × ¼–in.) or Plexiglas tube holder, 4°C ( appendix 1D)
  • Additional reagents and equipment for cell culture (unit 1.1) and gel electrophoresis (unit 6.1) or immunoprecipitation (unit 6.2)
NOTE: All solutions and equipment coming into contact with living cells must be sterile and appropriate aseptic techniques should be used accordingly.NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO 2 to maintain pH 7.4.

Alternate Protocol 1: Lysis of Cells by Boiling in SDS

  • SDS lysis buffer (see recipe)
  • RIPA correction buffer (see recipe)
  • Immunoprecipitate wash buffer (see recipe)
  • Fixed Staphylococcus aureus bacteria (Pansorbin, Calbiochem; optional)
  • Boiling water bath
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Figures

Videos

Literature Cited

Literature Cited
   Brugge, J.S. and Erikson, R.L. 1977. Identification of a transformation‐specific antigen induced by an avian sarcoma virus. Nature 269:346‐348.
   Chen, J., Stall, A.M., Herzenberg, L.A., and Herzenberg, L.A. 1990. Differences in glycoprotein complexes associated with IgM and IgD on normal murine B cells potentially enable transduction of different signals. EMBO J. 9:2117‐2124.
   Osman, N., Ley, S.C., and Crumpton, M.J. 1992. Evidence for an association between the T cell receptor/CD3 antigen complex and the CD5 antigen complex in human T lymphocytes. Eur. J. Immunol. 22:2995‐3000.
   Sefton, B.M., Hunter, T., and Beemon, K. 1980. Temperature‐sensitive transformation by Rous sarcoma virus and temperature‐sensitive protein kinase activity. J. Virol. 33:220‐229.
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