title: Single-Step Affinity Purification of ERK Signaling Complexes Using the Streptavidin-Binding Peptide (SBP) Tag.
abstract: Elucidation of biological functions of signaling proteins is facilitated by studying their protein-protein interaction networks. Affinity purification combined with mass spectrometry (AP-MS) has become a favorite method to study protein complexes. Here we describe a procedure for single-step purification of ERK (Rolled) and associated proteins from Drosophila cultured cells. The use of the streptavidin-binding peptide (SBP) tag allows for a highly efficient isolation of native ERK signaling complexes, which are suitable for subsequent analysis by mass spectrometry. Our analysis of the ERK interactome has identified both known and novel signaling components. This method can be easily adapted for SBP-based purification of protein complexes in any expression system.
authors.affiliation: Department of Biology, University of Massachusetts Boston, 100 Morrissey Blvd., Boston, MA, 02125, USA.
authors.forename: Liu
authors.initials: L
authors.lastname: Yang
authors.affiliation: Department of Biology, University of Massachusetts Boston, 100 Morrissey Blvd., Boston, MA, 02125, USA. Alexey.veraksa@umb.edu.
authors.forename: Alexey
authors.initials: A
authors.lastname: Veraksa
mesh_terms: D000818:Animals; D002352:Carrier Proteins; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D004330:Drosophila; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015870:Gene Expression; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics; D046912:Multiprotein Complexes / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
chemical_list: D002352:Carrier Proteins; D046912:Multiprotein Complexes; D011993:Recombinant Fusion Proteins; C452700:streptavidin-binding peptide; D048049:Extracellular Signal-Regulated MAP Kinases
keywords: Affinity purification; Drosophila; ERK; Mass spectrometry; SBP; Streptavidin-binding peptide