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Protein‐Protein Interactions Identified by Pull‐Down Experiments and Mass Spectrometry

The aim of this unit is to provide a method for the identification of new protein‐protein interactions. Pull‐down experiments with GST fusion proteins attached to glutathione beads are a screening technique for identification of protein‐protein interactions. When coupled with mass spectrometry, pull‐downs can be considered as the protein‐based equivalent of a yeast two‐hybrid screen. To improve the isolation of specific binding partners, pull‐down methods are described involving the use of cross‐linking, large‐scale tissue lysates, and spin columns. Alternative techniques are detailed for isolating activation state‐dependent protein interactions with small GTPases. Appropriate methods of sample preparation for mass spectrometry‐based identification of interacting proteins are described, including specialized gel staining techniques, band excision, and in‐gel tryptic digestion. Data interpretation and the most commonly encountered problems are discussed.