GST pull-down assay


A GST (glutathione-S-transferase) pull-down assay is a technique used to study protein-protein interactions in vitro. It involves the addition of a fusion tag for the GST enzyme (tag = 26kDa) at the N-terminus (5' end) of the gene encoding one of the proteins of interest. The GST-tagged protein can then be purified by virtue of its high affinity for glutathione beads, washed of contaminants, and eluted using competing glutathione which binds preferentially to the beads.

To demonstrate that protein X interacts with protein Y:

1. Create a GST-X fusion protein
2. Use glutathione-coated assay beads to purify GST-X
3. If GST-X binds Y then Y should also be present on the bead (use a Y-specific antibody to verify this)