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.
Brymora
Adam
A
Children's Medical Research Institute, Westmead NSW, Australia.
Valova
Valentina A
VA
Robinson
Phillip J
PJ
D000465:Algorithms; D000818:Animals; D003432:Cross-Linking Reagents / Q000494:pharmacology; D005978:Glutathione / Q000737:chemistry; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D008954:Models, Biological; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012685:Sepharose / Q000737:chemistry; D014020:Tissue Extracts / Q000737:chemistry / Q000302:isolation & purification
D003432:Cross-Linking Reagents; D011993:Recombinant Fusion Proteins; D014020:Tissue Extracts; D012685:Sepharose; D005982:Glutathione Transferase; D020559:Monomeric GTP-Binding Proteins; D005978:Glutathione