Fluorescent Proteins as Proteomic Probes*S
- From the ‡Laboratory for Mass Spectrometry and Gaseous Ion Chemistry and the §Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York 10021
- ¶To whom correspondence may be addressed: Laboratory for Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, 1230 York Ave., Box 170, New York, NY 10021. Tel.: 212-327-8847; Fax: 212-327-7547; E-mail: chait{at}mail.rockefeller.edu
Abstract
Protein complexes mediate the majority of cellular processes. Knowledge of the localization and composition of such complexes provides key insights into their functions. Although green fluorescent protein (GFP) has been widely applied for in vivo visualization of proteins, it has been relatively little used as a tool for the isolation of protein complexes. Here we describe the use of the standard GFP tag to both visualize proteins in living cells and capture their interactions via a simple immunoaffinity purification procedure. We applied this method to the analysis of a variety of endogenous protein complexes from different eukaryotic cells. We show that efficient isolations can be achieved in 5–60 min. This rapid purification helps preserve protein complexes close to their original state in the cell and minimizes nonspecific interactions. Given the wide use and availability of GFP-tagged protein reagents, the present method should greatly facilitate the elucidation of many cellular processes.
Footnotes
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Published, MCP Papers in Press, September 9, 2005, DOI 10.1074/mcp.M500227-MCP200
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↵1 The abbreviations used are: GFP, green fluorescent protein; FIA, Freund’s incomplete adjuvant; Ab, antibody; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; YFP, yellow fluorescent protein; NPC, nuclear pore complex.
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↵* This work was supported by National Institutes of Health Grants RR00862 (to B. T. C.), CA89810 (to B. T. C. and M. P. R.), GM062427 (to M. P. R.), RR022220 (to M. P. R. and B. T. C.), and by Women & Science fellowship Grant CEN5300379 (to I. M. C.).The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.
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↵‖ To whom correspondence may be addressed: Laboratory of Cellular and Structural Biology, The Rockefeller University, 1230 York Ave., Box 213, New York, NY 10021. Tel.: 212-327-8135; Fax: 212-327-7193; E-mail: rout{at}rockefeller.edu
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- Received July 21, 2005.
- Revision received September 2, 2005.
- The American Society for Biochemistry and Molecular Biology
