Many proteins function with partners or as components of a large multiprotein complex. Understanding these interactions is critical to our understanding of biological pathways and cellular function. Discovery of protein:protein interactions is accomplished via several different methods including yeast two hybrid, co-immunoprecipitation and tandem affinity purification (TAP). Using these methods to characterize protein interactions can be a time consuming and difficult process.
Glutathione-S Transferase (GST) pull-down is becoming an increasingly important tool for validation of suspected protein:protein interactions and also for identification of new interacting partners. GST pull-down uses a GST-fusion protein (bait) expressed typically in E.coli and then bound to glutathione (GSH)-coupled particles to affinity purify any proteins (prey) that interact with the bait from a pool of proteins in solution.
Prey proteins can be obtained from multiple sources including cell lysates, purified proteins and cell-free expression systems. Using cell free systems enables the researcher to easily express a variety of prey proteins and map the domain necessary for a successful interaction to occur.
A short animation illustrating this technique can be viewed at: http://www.promega.com/paguide/animation/selector.htm?coreName=tnt01
For examples of using this technique to define required protein domains or for the analysis of the effects of mutations refer to these publications:
Latest posts by Gary Kobs (see all)
- HaloTag Application: Fluorescence Under Stress - May 11, 2018
- Beer Is Complicated: Proteome Analysis via Mass Spectrometry - April 11, 2018
- Characterizing Multi-Subunit Protein Complexes Using Cell-Free Expression - March 12, 2018