Pull-down

GST pull-down is a classical in vitro protein interaction verification technique based on the high-affinity binding between Glutathione S-Transferase (GST) and Glutathione (GSH). A target protein is fused with GST and expressed, then immobilized onto GSH-coated magnetic beads or agarose resin to serve as the “bait”. Upon incubation with a lysate or purified protein containing candidate binding partners (the “prey”), specific interactions lead to capture and enrichment of the prey. After stringent washing to eliminate non-specific binding, the interacting proteins are identified via Western blot or mass spectrometry.

His-tag pull-down is an in vitro protein interaction technique based on Immobilized Metal Affinity Chromatography (IMAC), leveraging the high-affinity coordination between a hexahistidine tag (His₆) fused to the N- or C-terminus of a recombinant protein and transition metal ions (e.g., Ni²⁺, Co²⁺). The “bait” protein is immobilized onto Ni-NTA (nickel-nitrilotriacetic acid) or Co-NTA magnetic beads/agarose resin. Upon incubation with cell lysates, tissue extracts, or in vitro translation products containing candidate “prey” proteins, specific capture occurs. After washing away non-specific binders, the interaction complex is identified and analyzed.

Streptavidin-Biotin pull-down is an in vitro target fishing technique built upon the ultra-high affinity non-covalent interaction between Streptavidin and Biotin. Biotin serves as a small-molecule labeling probe, chemically conjugated to candidate drug molecules, metabolites, or ligands to form a “biotinylated bait”. After incubation with cell lysates, the bait is captured using streptavidin immobilized on agarose beads or magnetic beads. Given the extraordinary binding strength and stability, the complex remains intact even under stringent washing conditions (high salt, detergents, extreme pH), enabling efficient enrichment and identification of low-abundance interacting proteins.