Abstract
Affinity purification is a procedure based on the specific binding interactions between a ligand chemically coupled to a resin and a target molecule. A common application is the use of antibody as immobilized ligands. The purification of antigens by antibody-affinity chromatography is widely used to detect factors interacting with a protein of interest, and when coupled to mass spectrometry, it is a powerful approach for the identification of associated factors. In general, interacting proteins are isolated from cells expressing an epitope-tagged version of the protein of interest (1). Short peptides (tags) engineered at the amino- or carboxy-terminal end of proteins are quite useful for the purification of protein complexes through the use of tag-specific anti- body covalently coupled to a solid matrix. The use of tagged proteins is particularly effective for the isolation of native protein complexes from yeast cells, where these proteins can be expressed at their natural level (2– 4). On the other hand, it is difficult to express engineered proteins at physiological levels in mammalian cells. Because overexpression may alter the network of protein interactions, the purification by affin- ity to an engineered epitope tag may not provide a faithful representation of the factors that are associated with the endogenous protein. To overcome this potential problem, many investigators choose to employ an affinity purification procedure that uses an antibody that recognizes the endogenous protein. This approach has been valuable in the identification of protein complexes involved in gene transcription (5- 6) and DNA repair (7).
Similar content being viewed by others
References
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., et al. (2001) The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24, 218–229.
Honey, S., Schneider, B. L., Schieltz, D. M., Yates, J. R., and Futcher, B. (2001) A novel multiple affinity purification tag and its use in identification of proteins associated with a cyclin-CDK complex. Nucleic Acids Res. 29, E24.
Reisdorf, P., Maarse, A. C., and Daignan-Fornier, B. (1993) Epitope-tagging vectors designed for yeast. Curr. Genet. 23, 181–183.
Dynlacht, B. D., Hoey, T., and Tjian, R. (1991) Isolation of coactivators associated with the TATA-binding protein that mediate transcriptional activation. Cell 66, 563–576.
Comai, L., Tanese, N., and Tjian, R. (1992) The TATA-binding protein and associated factors are integral components of the RNA polymerase I transcription factor, SL1. Cell 68,965–976.
Wang, Y., Cortez, D., Yazdi, P., Neff, N., Elledge, S. J., and Qin, J. (2000) BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures. Genes Dev. 14, 927–939.
Comai, L., Zomerdijk, J. Beckmann, H., Zhou, S., Admon, A., and Tjian, R. (1994) Reconstitution of transcription factor SL1: exclusive binding of TBP by SL1 or TFIID subunits. Science 266, 1966–1972.
Pandey, A., Andersen, J. S., and Mann, M. (2000) Use of mass spectrometry to study signaling pathways. Sci. STKE 37, PL1.
Harlow, E., and Lane, D. (1999). Using Antibodies: A Laboratory Manual. (2nd edition), Cold Spring Harbor, Cold Spring Harbor, NY.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2005 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Navarro, S., Comai, L. (2005). Antibody- Affinity Purification to Detect Interacting Proteins. In: Walker, J.M. (eds) The Proteomics Protocols Handbook. Springer Protocols Handbooks. Humana Press. https://doi.org/10.1385/1-59259-890-0:683
Download citation
DOI: https://doi.org/10.1385/1-59259-890-0:683
Publisher Name: Humana Press
Print ISBN: 978-1-58829-343-5
Online ISBN: 978-1-59259-890-8
eBook Packages: Springer Protocols