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Single‐step Strep ‐tag® purification for the isolation and identification of protein complexes from mammalian cells
Author(s) -
Junttila Melissa R.,
Saarinen Susanna,
Schmidt Thomas,
Kast Juergen,
Westermarck Jukka
Publication year - 2005
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200400991
Subject(s) - affinity chromatography , protein phosphatase 2 , multiprotein complex , protein purification , identification (biology) , mass spectrometry , biology , elution , computational biology , proteomics , tandem affinity purification , immunoprecipitation , biochemistry , isolation (microbiology) , flag tag , cell culture , chemistry , chromatography , phosphatase , phosphorylation , bioinformatics , genetics , enzyme , recombinant dna , gene , botany , fusion protein
Identification of protein complexes is the key to understanding cellular functions. In this study, we present a novel method for the identification of multiprotein complexes from mammalian cells. By using the Strep ‐tag affinity chromatography method, enabling fast and simple one‐step purification, coupled with competitive elution under physiological conditions, we successfully purified a PP2A holoenzyme protein complex from a cultured mammalian cancer cell line. We identified, by mass spectrometry, both known and novel interacting proteins for PP2A, and demonstrate that the purified PP2A complex is functional. The benefits and potential applications of the Strep ‐tag method for protein complex purification are discussed.