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Molecular & Cellular Proteomics 4:1933-1941, 2005.
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
Research
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
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.
¶ 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
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