Sponsored,vendor-submitted protocol Published in April 2009
Many proteins function with partners or as components of large multi-protein complexes. Understanding these interactions is critical to understanding biological pathways and cellular function. Glutathione-S Transferase (GST) pull-down (1) is an important tool for validation of suspected protein:protein interactions and for identification of interacting partners (2–5). In GST pull-down assays a GST-fusion bait protein is expressed in E. coli, then bound to glutathione (GSH)-coupled particles and used to affinity purify any proteins (prey) that interact with the bait from a pool of proteins in solution (Figure 1).
Prey proteins come from multiple sources including cell lysates, purified proteins and cell-free expression systems. Cell-free systems easily express a variety of prey proteins and map the domain necessary for a successful interaction.
Figure 1: Schematic of pull-down assay using bacterial expression of bait protein and cell-free expression for the prey protein.
