title: Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells.
abstract: The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.
authors.affiliation: Department of Microbiology, The University of Texas Southwestern Medical Center.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Ma
authors.affiliation: Department of Microbiology, The University of Texas Southwestern Medical Center.
authors.forename: Victor
authors.initials: V
authors.lastname: Fung
authors.affiliation: Department of Microbiology, The University of Texas Southwestern Medical Center; Ivan.Dorso@utsouthwestern.edu.
authors.forename: Iván
authors.initials: I
authors.lastname: D'Orso
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods; D056906:Cyclin T / Q000235:genetics / Q000302:isolation & purification; D042863:Cyclin-Dependent Kinase 9 / Q000235:genetics / Q000302:isolation & purification; D000939:Epitopes / Q000737:chemistry; D005057:Eukaryotic Cells / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D046912:Multiprotein Complexes / Q000737:chemistry / Q000302:isolation & purification*; D042925:Positive Transcriptional Elongation Factor B / Q000302:isolation & purification; D060066:Protein Interaction Maps; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*
chemical_list: C503684:CCNT1 protein, human; D056906:Cyclin T; D000939:Epitopes; D046912:Multiprotein Complexes; D011506:Proteins; D049452:Green Fluorescent Proteins; D042925:Positive Transcriptional Elongation Factor B; C467969:CDK9 protein, human; D042863:Cyclin-Dependent Kinase 9