TwoHybridExperimenten

Two-Hybrid Experiments are a set of molecular biology methods designed to detect protein–protein interactions in vivo by reconstituting a functional transcription factor from two separate domains. The most widely used version is the yeast two-hybrid (Y2H) assay, introduced by Fields and Song in 1989, which popularized the concept. The principle is that a protein of interest, called the bait, is fused to a DNA-binding domain, while a second protein, called the prey, is fused to a transcriptional activation domain. If bait and prey interact, the two domains are brought together and drive expression of a reporter gene in the host cell.

Common reporters include HIS3, ADE2, or lacZ in yeast, providing growth selection or colorimetric readouts. Yeast cells are the traditional host, but variants exist for mammalian cells (mammalian two-hybrid) or bacteria (bacterial two-hybrid) to accommodate different cellular contexts and post-translational modifications. High-throughput versions enable large-scale screening to map interaction networks.

Typical workflow involves cloning the two proteins into compatible bait and prey vectors, introducing them into the host, and selecting for reporter activation. Positive interactions are further tested with orthogonal assays such as co-immunoprecipitation or co-localization, to validate the findings.

Limitations include false positives caused by auto-activation or non-specific interactions and false negatives due to improper folding, mislocalization, or missing cofactors. Interactions observed in two-hybrid assays often require independent confirmation and may be context-dependent. Despite caveats, two-hybrid experiments have become a foundational approach for mapping protein interaction networks and inferring biological function.