Identifying nuclear protein-protein interactions using GFP affinity purification and SILAC-based quantitative mass spectrometry

H Irem Baymaz; Cornelia G Spruijt; Michiel Vermeulen

doi:10.1007/978-1-4939-1142-4_15

Identifying nuclear protein-protein interactions using GFP affinity purification and SILAC-based quantitative mass spectrometry

Methods Mol Biol. 2014:1188:207-26. doi: 10.1007/978-1-4939-1142-4_15.

Authors

H Irem Baymaz¹, Cornelia G Spruijt, Michiel Vermeulen

Affiliation

¹ Department of Molecular Cancer Research, University Medical Center Utrecht, 85060, 3508 AB, Utrecht, The Netherlands.

PMID: 25059614
DOI: 10.1007/978-1-4939-1142-4_15

Abstract

Many cellular proteins assemble into macromolecular protein complexes. Therefore, identifying protein-protein interactions (PPIs) is essential to gain insight into the function of proteins. Recently established quantitative mass spectrometry-based techniques have significantly improved the unbiased search for PPIs. In this chapter, we describe a single-step GFP affinity purification method combined with SILAC-based quantitative mass spectrometry that can be used to identify nuclear PPIs in mammalian cells.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acids / chemistry*
Cell Line
Cell Nucleus / metabolism
Chromatography, Affinity
Green Fluorescent Proteins / metabolism*
Isotope Labeling / methods*
Mass Spectrometry / methods*
Nuclear Proteins / chemistry*
Nuclear Proteins / genetics
Nuclear Proteins / isolation & purification
Nuclear Proteins / metabolism*
Peptides / isolation & purification
Peptides / metabolism
Protein Interaction Mapping / methods*
Proteolysis
Transfection

Substances

Amino Acids
Nuclear Proteins
Peptides
Green Fluorescent Proteins