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.
Highlights
Protein complexes mediate the majority of cellular processes
5) The isolated proteins are eluted from the magnetic beads and resolved by one-dimensional gel electrophoresis. 6) The purified proteins are identified by MALDI mass spectrometry
We have demonstrated the applicability of this method to the analysis of protein complexes located in various subcellular compartments and have isolated even low abundance proteins in both yeast and mammalian systems
Summary
Protein complexes mediate the majority of cellular processes. Knowledge of the localization and composition of such complexes provides key insights into their functions. 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. The use of multiple tags, such as the two-step tandem affinity purification tag strategy, has become popular [8] All these tags have proved highly effective for the isolation of protein complexes, they cannot be readily visualized in living cells. We describe how the use of GFP to both visualize proteins in live cells and capture their interactions via immunoaffinity purification on magnetic beads promises to facilitate our understanding of the temporal and spatial dynamics of protein interactions
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