Fluorescent proteins as proteomic probes.
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.
Cristea
Ileana M
IM
Laboratory for Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, New York 10021, USA.
Williams
Rosemary
R
Chait
Brian T
BT
Rout
Michael P
MP
D000818:Animals; D000906:Antibodies; D002460:Cell Line; D002846:Chromatography, Affinity; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D008164:Luminescent Proteins / Q000737:chemistry*; D016366:Open Reading Frames; D020543:Proteome / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013205:Staphylococcal Protein A / Q000737:chemistry
D000906:Antibodies; D007074:Immunoglobulin G; D008164:Luminescent Proteins; D020543:Proteome; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D049452:Green Fluorescent Proteins