Two-hybrid systems are extremely 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 at the TATA box and increase transcription.
In the CheckMate™ Mammalian Two-Hybrid System the DNA-binding domain and the transcriptional activation domain, produced by separate plasmids, are closely associated when one protein ("X") fused to a DNA-binding domain interacts with a second protein ("Y") fused to a transcriptional activation domain. In this system, interaction between proteins X and Y results in transcription of a reporter gene.
For more information, see the Protocols & Applications Guide.
- Confirming suspected interactions between two proteins and identifying domains/residues involved in protein:protein interactions.
- Sadowski, I. et al. (1988) Nature 335, 563–4.