UNIT 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry
Published Online: 1 MAY 2004
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Cell Biology
How to Cite
Brymora, A., Valova, V. A. and Robinson, P. J. 2004. Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry. Current Protocols in Cell Biology. 22:17.5:17.5.1–17.5.51.
- Published Online: 1 MAY 2004
- Published Print: MAR 2004
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.
- protein-protein interactions;
- small GTPase;
- GST fusion protein;
- mass spectrometry;
- spin column;
- in-gel digestion;
- tissue lysate