Unit

UNIT 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry

  1. Adam Brymora,
  2. Valentina A. Valova,
  3. Phillip J. Robinson

Published Online: 1 MAY 2004

DOI: 10.1002/0471143030.cb1705s22

Current Protocols in Cell Biology

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.

Author Information

  1. Children's Medical Research Institute, Westmead NSW, Australia

Publication History

  1. Published Online: 1 MAY 2004
  2. Published Print: MAR 2004

Abstract

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.

Keywords:

  • Pull-down;
  • protein-protein interactions;
  • small GTPase;
  • GST fusion protein;
  • mass spectrometry;
  • spin column;
  • cross-linking;
  • in-gel digestion;
  • tissue lysate