The two-hybrid method facilitates the discovery of protein–protein interactions. How does this technique work? Can you think of reasons for obtaining a false-positive result, that is, where the proteins encoded by two clones interact in the two-hybrid system but do not interact in the organism in which they naturally occur? Can you think of reasons you might obtain a false-negative result, in which the two proteins interact in vivo but fail to interact in the two-hybrid system?

Key Concepts

Two-Hybrid System

The two-hybrid system is a molecular biology technique used to detect protein-protein interactions within a cell. It involves the use of two fusion proteins: one with a DNA-binding domain and the other with a transcriptional activation domain. When the two proteins interact, they bring these domains together, activating the transcription of a reporter gene, which can be measured. This method is crucial for understanding cellular processes and identifying potential interactions between proteins.

False Positives

False positives in the two-hybrid system occur when the assay indicates an interaction between two proteins that does not exist in vivo. This can happen due to non-specific binding, where proteins interact in the artificial environment of the assay but not in their natural context. Other reasons include the overexpression of proteins, which can lead to interactions that are not physiologically relevant, or the presence of other cellular factors that may influence the assay's outcome.

False Negatives

False negatives occur when the two-hybrid system fails to detect an interaction that actually occurs in vivo. This can result from several factors, such as the proteins being expressed at levels too low to detect, or the requirement of post-translational modifications that are not replicated in the assay. Additionally, the cellular environment in which the proteins naturally interact may provide necessary cofactors or conditions that are absent in the two-hybrid system, leading to a lack of detectable interaction.