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Detection of Protein‐Protein Interactions by Coprecipitation

Elaine A. Elion¹

¹Harvard Medical School, Boston, Massachusetts

Publication Name:

Current Protocols in Protein Science

Unit Number:

Unit 19.4

DOI:

10.1002/0471140864.ps1904s49

Online Posting Date:

August, 2007

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Abstract

Co-precipitation of proteins from whole cell extracts is a valuable approach to test for physical interactions between proteins of interest. It may be the method of choice, or may be used in conjunction with other procedures to detect protein-protein interactions, such as two-hybrid analyis and co-purification schemes, and tests of physical associations using purified proteins. Curr. Protoc. Protein Sci. 49:19.4.1-19.4.10. © 2007 by John Wiley & Sons, Inc.

Keywords: co-immunoprecipitation; immunoprecipitation; protein interactions; protein tag; antibody; immunoblot

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Unit Introduction
Strategic Planning
Basic Protocol: Coprecipitating Proteins with Protein A/G–Sepharose
Alternate Protocol: Coprecipitating a GST Fusion Protein
Reagents and Solutions
Commentary
Bibliography
Figures

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Materials

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

Materials

Whole-cell extract (see Strategic Planning)
Antibody against protein or epitope tag of interest
5 M NaCl
Co-immunoprecipitation buffer (see recipe)
Protein A/G–Sepharose slurry (see recipe)
2× sample buffer for SDS-PAGE (unit 10.1)

Test tube rotator
20-ml syringe and 18-G needle
Hamilton syringe

Additional reagents and equipment for SDS-PAGE (unit 10.1) and immunoblotting (unit 10.10)

Alternate Protocol: Coprecipitating a GST Fusion Protein

Additional Materials (also see Basic Protocol)

Glutathione-agarose or glutathione-Sepharose slurry (see recipe)

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Figures

Figure 19.4.1

Flowchart for the coprecipitation of two proteins that have been differentially tagged and introduced into the host organism. Ig h and Ig l, immunoglobulin heavy and light chains; NT, no tag.

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Literature Cited

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.
	Coligan, J.E., Bierer, B.E., Margulies, D.H., Shevach, E.M., and Strober, W. (eds). 2007. Current Protocols in Immunology. John Wiley & Sons, Hoboken, N.J.
	Feng, Y., Song, L.-Y., Kincaid, E., Mahanty, S.K., and Elion, E.A. 1998. Functional binding between G 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. 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
	Harlow, E. and Lane, D. 1998. Using Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
	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.
	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.
	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.
	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.
	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.
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.