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Pull-Down and Two-Hybrid Systems

Pull-down assays and two-hybrid systems are commonly used methods for studying protein interactions. We offer pulldown systems based on GST or HaloTag® affinity tags, and a two-hybrid system designed for identifying mammalian protein interactions.

The MagneGST™ Pull-Down Systems provide GSH-linked magnetic particles that allow simple immobilization of GST-fusion bait proteins from bacterial lysates, together with an in vitro transcription/translation system for expression of prey proteins. In the HaloTag® Mammalian Pull-Down System, the protein of interest is bound to an affinity resin using HaloTag® protein, which binds to the resin rapidly, covalently and irreversibly. These properties increase the chance of capturing protein complexes and retaining them after capture.

In the CheckMate™ Mammalian Two-Hybrid System, one protein is fused to a DNA-binding domain and a second protein is fused to a transcriptional activation domain. Interaction between the proteins results in transcription of a reporter gene, resulting in bioluminescence-based detection of interacting protein partners.

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What are Pull-Down and Two-Hybrid Systems?

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.