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 Molecular Biology
Unit Number:  Unit 18.2
DOI:  10.1002/0471142727.mb1802s40
Online Posting Date:  May, 2001
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Abstract

This unit describes 32Pi labeling and lysis of cultured cells to be used for subsequent immunoprecipitation of proteins. The approach is appropriate, however, for labeling any cellular constituent with 32P. This procedure is suitable for insect, avian, and mammalian cells and can be used with both adherent and nonadherent cultures. The general approach involves biosynthetic labeling with 32Pi in medium containing a reduced concentration of phosphate. This approach can also be modified to label any cellular constituents with 32Pi. The first procedure described is 32Pi labeling of adherent or nonadherent (e.g., hematopoietic) cells with subsequent lysis in a detergent buffer. More rigorous lysis conditions to be used for working with proteins that are difficult to solubilize are also described.

     
 
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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 3F) supplemented with the usual concentration of serum or serum dialyzed against phosphate‐free saline, 37°C
  • 1 Ci/ml H 332PO 4 in HCl (carrier free ICN)
  • recipeTris‐buffered or recipephosphate‐buffered saline ( recipeTBS or recipePBS; see reciperecipes), cold
  • recipeMild lysis buffer or recipeRIPA lysis buffer (see reciperecipes)
  • 1‐in.‐thick Plexiglas shield ( appendix 1A )
  • Plugged, aerosol‐resistant pipet tips
  • Plexiglas box ( appendix 1A ), 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 1A)
  • Additional reagents and equipment for cell culture (see Chapter , introduction) and gel electrophoresis (unit 10.2), immunoprecipitation (unit 10.16), or protein purification (units 10.9 10.11)
NOTE: All culture incubations are performed in a humidified 37°C, 10% CO 2 incubator unless otherwise specified.

Alternate Protocol 1: Lysis of Cells by Boiling in SDS

  • recipeSDS lysis buffer (see recipe)
  • recipeRIPA correction buffer (see recipe)
  • recipeImmunoprecipitate 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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