ABSTRACT
In vivo transcription-based assays for protein-protein interactions such as the two-hybrid system are powerful methods for identifying novel proteins based on their physical association with known proteins of biological interest, or for characterizing the degree and nature of interactions between sets of proteins. Because of the complexity inherent in assays taking place within a living organism, a key issue for the effective use of two-hybrid approaches is the ability to determine whether apparent interactions are likely to be physiologically relevant.
In this chapter, a number of the different two-hybrid systems currently available for use will be reviewed. Then, taking as a model one such system, the Interaction Trap, examples of different reagents for use in varying the affinity range of detectable interactions will be outlined. Also set forth are a number of protocols to establish an appropriate set of conditions for either screening a library or analysing the interaction phenotype between protein sets. Finally, a number of general guidelines are suggested for trouble-shooting two-hybrid results, and for eliminating falsely positive interactions.
