Isolation of Proteins and Protein Complexes by Immunoprecipitation

  • Barbara Kaboord
  • Maria Perr
Part of the Methods in Molecular Biology™ book series (MIMB, volume 424)


Immunoprecipitation (IP) uses the specificity of antibodies to isolate target proteins (antigens) out of complex sample mixtures. Three different approaches for performing IP will be discussed; traditional (classical) method, oriented affinity method and direct affinity method. The traditional method of incubating the IP antibody with the sample and sequentially binding to Protein A or G agarose beads (resin) facilitates the most efficient target antigen recovery. However, this approach results in the target protein becoming contaminated with the IP antibody that can interfere with downstream analyses. The orientated affinity method uses Protein A or G beads to serve as an anchor to which the IP antibody is crosslinked thereby preventing the antibody from co-eluting with the target protein. Similarly, the direct affinity method also immobilizes the IP antibody except in this case it is directly attached to a chemically activated support. Both methods prevent co-elution of the IP antibody enablings reuse of the immunomatrix. All three approaches have unique advantages and can also be used for co-immunoprecipitation to study protein:protein interactions and investigate the functional proteome.

Key Words

Aldehyde activated support antibody contamination co-immunoprecipitation immune complex immunoaffinity support immunoprecipitation Protein A Protein G protein-protein interactions protein purification 



The authors would like to thank Dr. Ling Ren and Daryl Emery for valuable experimental contributions during method development.


  1. 1.
    Prinetti, A., Prioni, S., Chigorno, V., Karagogeos, D., Tettamanti, G. and Sonnino. S. (2001) Immunoseparation of sphingolipid-enriched membrane domains enriched in Src family protein tyrosine kinases and in the neuronal adhesion molecule TAG-1 by anti-GD3 ganglioside monoclonal antibody. J. Neurochem. 78,1162–7.CrossRefPubMedGoogle Scholar
  2. 2.
    Sargsyan, E., Baryshev, M., Szekely, L., Sharipo, A. and Mkrtchian, S.(2002) Identification of ERp29, an endoplasmic reticulum lumenal protein, as a new member of the thyroglobulin folding complex. J. Biol. Chem. 277, 17009–15.CrossRefPubMedGoogle Scholar
  3. 3.
    Heegaard, N. H., Hansen, M. Z., Sen, J. W., Christiansen, M. and Westermark, P. (2006) Immunoaffinity chromatographic and immunoprecipitation methods combined with mass spectrometry for characterization of circulating transthyretin. J. Sep. Sci 29, 371–7.CrossRefPubMedGoogle Scholar
  4. 4.
    Fujita, N., Kaito, M., Tanaka, H., Horiike, S., Urawa, N., Sugimoto, R., Konishi, M., Watanabe, S. and Adachi, Y.(2006) Hepatitis C virus free-virion and immune-complex dynamics during interferon therapy with and without ribavirin in genotype-1b chronic hepatitis C patients. J. Viral Hepat 13,190–8.CrossRefPubMedGoogle Scholar
  5. 5.
    Goedert, J.J., Rabkin, C. S. and Ross, S. R. (2006) Prevalence of serologic reactivity against four strains of mouse mammary tumor virus among US women with breast cancer. Br. J. Cancer 94, 548–51.CrossRefPubMedGoogle Scholar
  6. 6.
    Rascon, A., Lindgren, S., Stavenow, L., Belfrage, P., Andersson, K. E., Manganiello, V.C. and Degerman, E., (1992) Purification and properties of the cGMP-inhibited cAMP phosphodiesterase from bovine aortic smooth muscle. Biochim. Biophys. Acta. 1134,149–56.CrossRefPubMedGoogle Scholar
  7. 7.
    Stam, H. and Hulsmann, W. C. (1984) Effects of hormones, amino acids and specific inhibitors on rat heart heparin-releasable lipoprotein lipase and tissue neutral lipase activities during long-term perfusion. Biochim. Biophys. Acta. 794, 72–82.PubMedGoogle Scholar
  8. 8.
    Doppler, H., Storz, P., Li, J., Comb, M.J., and Toker, A. (2005) A phosphorylation state-specific antibody recognizes Hsp27, a novel substrate of protein kinase D. J. Biol. Chem. 280, 15013–19.CrossRefPubMedGoogle Scholar
  9. 9.
    Grønborg, M., Kristiansen, T.Z., Stensballe, A., Andersen, J.S., Ohara, O., Mann, M., Jensen, O.N., and Pandey, A. (2002) A mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phospho-specific antibodies. Mol. Cell. Proteomics 1, 517–27.CrossRefPubMedGoogle Scholar
  10. 10.
    Schneider, R., Bannister, A.J., Myers, F.A., Thorne, A.W., Crane-Robinson, C., and Kouzarides, T. (2004) Histone H3 lysine 4 methylation patterns in higher eukaryotic genes. Nature Cell Biol. 6, 73–7.CrossRefPubMedGoogle Scholar
  11. 11.
    Monferran, S., Paupert, J., Dauvillier, S., Salles, B., and Muller, C. (2004) The membrane form of the DNA repair protein Ku interacts at the surface with metalloproteinase 9. EMBO J. 23, 3758–68.CrossRefPubMedGoogle Scholar
  12. 12.
    Marcora, E., Gowan, K., and Lee, J.E. (2003) Stimulation of NeuroD activity by huntingtin and huntingtin-associated proteins HAP1 and MLK2. Proc. Natl. Acad. Sci. USA 100, 9578–83.CrossRefPubMedGoogle Scholar
  13. 13.
    Harlow, E. and Lane, D. (ed.) (1999). Using Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.Google Scholar
  14. 14.
    Schilling, B., Murray, J., Yoo, C. B., Row, R. H., Cusack, M. P,, Capaldi, R. A. and Gibson. B. W. (2006) Proteomic analysis of succinate dehydrogenase and ubiquinol-cytochrome c reductase (Complex II and III) isolated by immunoprecipitation from bovine and mouse heart mitochondria. Biochim. Biophys. Acta 1762, 213–22.PubMedGoogle Scholar
  15. 15.
    Gridley, S., Lane, W. S., Garner, C. W. and Lienhard, G. E. (2005) Novel insulin-elicited phosphoproteins in adipocytes. Cell Signal. 17, 59–66.CrossRefPubMedGoogle Scholar
  16. 16.
    Barnouin, K. (2004) Two-dimensional gel electrophoresis for analysis of protein complexes. Methods Mol. Biol 261, 479–98.PubMedGoogle Scholar
  17. 17.
    Faber, E. S., Sedlak, P., Vidovic, M. and Sah, P. (2006) Synaptic activation of transient receptor potential channels by metabotropic glutamate receptors in the lateral amygdala. Neuroscience 137, 781–94.CrossRefPubMedGoogle Scholar
  18. 18.
    Anzai, N., Miyazaki, H., Noshiro, R., Khamdang, S., Chairoungdua, A., Shin, H. J., Enomoto, A., Sakamoto, S., Hirata, T., Tomita, K., Kanai, Y. and Endou, H.(2004) The multivalent PDZ domain-containing protein PDZK1 regulates transport activity of renal urate-anion exchanger URAT1 via its C terminus. J. Biol Chem. 279, 45942–50.CrossRefGoogle Scholar
  19. 19.
    Qoronfleh, M. W., Ren, L., Emery, D., Perr, M. and Kaboord, B. (2003) Use of immunomatrix methods to improve protein–-protein interaction detection. J. Biomed. Biotechnol. 2003, 291–8.CrossRefPubMedGoogle Scholar
  20. 20.
    Sisson, T.H. and Castor, C.W. (1990). An improved method for immobilizing IgG antibodies on protein A-agarose. J. Immunol. Meth. 127, 215–20.CrossRefGoogle Scholar
  21. 21.
    Khundmiri, S. J., Rane, M. J., Lederer, E. D. (2003) Parathyroid hormone regulation of type II sodium-phosphate cotransporters is dependent on an A kinase anchoring protein. J. Biol. Chem 278, 10134–41.CrossRefPubMedGoogle Scholar
  22. 22.
    Domen, P. L., Nevens, J. R., Mallia, A. K., Hermanson, G. T. and Klenk, D. C. (1990) Site-directed immobilization of proteins. J. Chromatogr 510, 293–302.CrossRefPubMedGoogle Scholar
  23. 23.
    Hermanson, G.T., Mallia, A.K., and Smith, P. K. (ed.) (1992). Immobilized Affinity Ligand Techniques. Academic Press, Inc., San Diego, CA.Google Scholar
  24. 24.
    Seko, Y., Azmi, H., Fariss, R. and Ragheb, J. A. (2004) Selective cytoplasmic translocation of HuR and site-specific binding to the interleukin-2 mRNA are not sufficient for CD28-mediated stabilization of the mRNAJ Biol Chem. 279, 33359–67.CrossRefPubMedGoogle Scholar
  25. 25.
    Chen, Y. H. and Lu, Q. (2003) Association of nonreceptor tyrosine kinase c-yes with tight junction protein occludin by coimmunoprecipitation assay. Methods Mol. Biol. 218, 127–32.PubMedGoogle Scholar
  26. 26.
    Ransone, L. J. (1995) Detection of protein-protein interactions by coimmunoprecipitation and dimerization. Methods Enzymol. 254, 491–7.CrossRefPubMedGoogle Scholar
  27. 27.
    Li, Z., Zou, C. B., Yao, Y., Hoyt, M. A., McDonough, S., Mackey, Z. B., Coffino, P. and Wang, C. C.(2002) An easily dissociated 26 S proteasome catalyzes an essential ubiquitin-mediated protein degradation pathway in Trypanosoma brucei. J. Biol. Chem. 277, 15486–98.CrossRefPubMedGoogle Scholar
  28. 28.
    Loven, M. A., Muster, N., Yates, J. R.and Nardulli A. M. A novel estrogen receptor alpha-associated protein, template-activating factor Ibeta, inhibits acetylation and transactivation. Mol. Endocrinol. 17, 67–78.Google Scholar
  29. 29.
    Brymora, A., Cousin, M.A., Roufogalis, B.D., and Robinson, P.J. (2001) Enhanced protein recovery and reproducibility from pull-down assays and immunoprecipitations using spin columns. Anal. Biochem. 295, 119–22.CrossRefPubMedGoogle Scholar
  30. 30.
    Howell, J.M., Winstone, T.L., Coorssen, J.R., and Turner, R.J. (2006) An evaluation of in vitro protein-protein interaction techniques: Assessing contaminating background proteins. Proteomics 6, 2050–69.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Barbara Kaboord
    • 1
  • Maria Perr
    • 1
  1. 1.Pierce Biotechnology, Inc.RockfordIllinois

Personalised recommendations