Pull-down assays and two-hybrid systems are tools used to identify whether two or more proteins interact. These protein interaction assays can be used to confirm protein interactions identified by other means (such as co-immunoprecipitation assays) or to discover and identify new interacting protein partners.
In pull-down assays, a “bait” protein is immobilized on a resin using an affinity tag such as HaloTag or GST. “Prey” proteins that bind to the immobilized prey proteins are then captured from cell lysates or in vitro-expressed products. The pull-down assay protocol is divided into three phases: 1) the prey protein is expressed; 2) the bait protein is immobilized; and 3) the prey protein is added to the system and captured through bait-prey interaction.
Two-hybrid systems are powerful methods for detecting protein:protein interactions in vivo. The basis of two-hybrid systems is the modular domains found in some transcription factors: a DNA-binding domain, which binds to a specific DNA sequence, and a transcriptional activation domain, which interacts with the basal transcriptional machinery. A transcriptional activation domain in association with a DNA-binding domain will promote the assembly of RNA polymerase II complexes and increase transcription. In two-hybrid systems, interaction between protein partners brings the DNA binding and transcriptional activation domains into close proximity, promoting expression of a reporter gene.
Two-hybrid systems and in vitro pull-down assays can be used for genome- or proteome-wide identification of protein:protein interactions. In vitro protein pull-down assays can be performed using cell lysates, in vitro-expressed lysates, tissue samples, etc. These options are not possible with two-hybrid approaches.