Detection of Protein‐Protein Interactions by Coprecipitation

Elaine A. Elion1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 8.7
DOI:  10.1002/0471142727.im0807s76
Online Posting Date:  February, 2007
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Abstract

Coprecipitation of proteins from whole‐cell extracts is a valuable approach to test for physical interactions between proteins of interest. This unit describes basic approaches to immunoprecipitating tagged proteins from whole‐cell extracts. The approaches described can be adapted for other systems. In a typical experiment, as described here, cells are lysed and a whole‐cell extract is prepared under nondenaturing conditions. The protein is precipitated from the lysate with a solid‐phase affinity matrix, and the precipitate is tested for the presence of a second specifically associated protein. The approach can be used for native or epitope‐tagged proteins for which antibodies are available, or for recombinant proteins that have been engineered to bind with high affinity to a molecule that can be coupled to a solid‐phase matrix.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Coprecipitating Proteins with Protein A/G–Sepharose
  • Alternate Protocol 1: Coprecipitating a GST Fusion Protein
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Coprecipitating Proteins with Protein A/G–Sepharose

  Materials
  • Whole‐cell extract (see )
  • Antibody against protein or epitope of interest
  • 5 M NaCl
  • Co‐immunoprecipitation buffer (see recipe)
  • Protein A/G–Sepharose slurry (see recipe)
  • 2× sample buffer for SDS‐PAGE (unit 8.4)
  • Test tube rotator
  • 20‐ml syringe and 18‐G needle
  • Hamilton syringe
  • Additional reagents and equipment for SDS‐PAGE (unit 8.4) and immunoblotting (unit 8.10)

Alternate Protocol 1: Coprecipitating a GST Fusion Protein

  • Glutathione‐agarose or glutathione‐Sepharose slurry (see recipe)
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Figures

Videos

Literature Cited

   Auerbach, D., Galeuchet‐Schenk, B., Hottiger, M.O., and Stagliar, I. 2002. Genetic approaches to the identification of interactions between membrane proteins in yeast. J. Recept. Signal Transduct. Res. 22:471‐481.
   BioSupplyNet Source Book. 2005. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Bornhorst, J.A. and Falke, J.J. 2000. Purification of proteins using polyhistidine affinity tags. Methods Enzymol. 326:245‐254.
   Dunn, B. and Wobbe, C.R. 2003. Preparation of protein extracts from yeast. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 13.13.1‐13.13.9. John Wiley & Sons, Hoboken, N.J.
   Feng, Y., Song, L.‐Y., Kincaid, E., Mahanty, S.K., and Elion, E.A. 1998. Functional binding between Gβ and the LIM domain of Step is required to activate the MEKK Ste11. Curr. Biol. 8:267‐278.
   Field, J., Nikawa, J., Broek, D., MacDonald, B., Rodgers, L., Wilson, I.A., Lerner, R.A., and Wigler, M. 1988. Purification of RAS‐responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol. Cell. Biol. 8:2159‐2165.
   Gavin, A. and Superti‐Furga, G. 2003. Protein complexes and proteome organization from yeast to man. Curr. Opin. Chem. Biol. 7:21‐27.
   Harlow, E. and Lane, D. 1998. Using Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Kerppola, T.K. 2006. Complementary methods for studies of protein interactions in living cells. Nat. Methods 3:969‐971.
   Knappik, A. and Pluckthun, A. 1994. An improved affinity tag based on the FLAG peptide for the detection and purification of recombinant antibody fragments. Biotechniques 17:754‐761.
   Kolodziej, P.A. and Young, R.A. 1991. Epitope tagging and protein surveillance. Methods Enzymol. 194:508‐519.
   Murphy, C.I., Piwnica‐Worms, H., Grünwald, S., Romanow, W.G., Francis, N., and Fan, H.‐Y. 2003. Expression and purification of recombinant proteins using the baculovirus system. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.11.1‐16.11.15. John Wiley & Sons, Hoboken, N.J.
   Nguyen, T.N. and Goodrich, J.A. 2006. Protein‐protein interaction assay: Eliminating false‐positive interactions. Nat. Methods 3:135‐139.
   Phizicky, E.M. and Fields, S. 1995. Protein‐protein interactions: Methods for detection and analysis. Microbiol. Rev. 59:94‐123.
   Puig, O., Caspary, F., Rigaut, G., Rutz, B., Bouveret, E., Bragado‐Nilsson, E., Wilm, M., and Seraphin, B. 2001. The tandem affinity purification (TAP) method: A general procedure of protein complex purification. Methods 24:218‐229.
   Riggs, P. 2003. Expression and purification of maltose‐binding protein fusions. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.6.1‐16.6.14. John Wiley & Sons, Hoboken, N.J.
   Skerra, A. and Schmidt, T.G.M. 2005. Use of the Strep‐tag and streptavidin for detection and purification of recombinant proteins. Methods Enzymol. 326:271‐304.
   Smith, D.B. and Corcoran, L.M. 2003. Expression and purification of glutathione‐S‐transferase fusion proteins. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 16.7.1‐16.7.13. John Wiley & Sons, Hoboken, N.J.
   Toby, G.G. and Golemis, E.A. 2001. Using the yeast interaction trap and other two‐hybrid‐based approaches to study protein‐protein interactions. Methods 24:201‐217.
   Treco, D.A. and Winston, F. 2003. Growth and manipulation of yeast. In Current Protocols in Molecular Biology F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 13.2.1‐13.2.12. John Wiley & Sons, Hoboken, N.J.
   Tyers, M., Tokiwa, G., and Futcher, A.B. 1993. Comparison of the Saccharomyces cerevisiae G1 cyclins: Cln3 may be an upstream activator of Cln1, Cln2, and other cyclins. EMBO J. 11:1773‐1784.
   Wang, Y., Chen, W., Simpson, D.M., and Elion, E.A. 2005. Cdc24 regulates nuclear shuttling and recruitment of the Ste5 scaffold to a heterotrimeric G protein in Saccharomyces cerevisiae. J. Biol. Chem. 280:13084‐13096.
   Witzgall, R., O'Leary, E., and Bonventre, J.V. 1994. A mammalian expression vector for the expression of GAL4 fusion proteins with an epitope tag and histidine tail. Anal. Biochem. 2:291‐298.
   Yang, P., Sampson, H.M., and Krause, H.M. 2006. A modified tandem affinity purification strategy identifies cofactors of the Drosophila nuclear receptor dHNF4. Proteomics 6:927‐935.
   Zhang, N. and Chen, J.L. 2005. Purification of recombinant proteins and study of protein interaction by epitope tagging. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 10.15.1‐10.15.10. John Wiley & Sons, Hoboken, N.J.
Key References
   BioSupplyNet Source Book, 2005. See above.
  Published yearly. Instant access is available at http://www.biosupplynet.com.
   Harlow and Lane, 1998. See above.
  General discussion of immunoprecipitation techniques and reagents.
   Phizicky and Fields, 1995. See above.
  General discussion of methodologies for detecting protein‐protein interactions as well as their merits and drawbacks.
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