Mammalian Two-Hybrid Assay for Detecting Protein-Protein Interactions in Vivo
The mammalian two-hybrid system is a very powerful tool to investigate protein-protein interactions in terms of functional domains and identify potential binding ligands and partners of a protein. Compared with the yeast two-hybrid system, the mammalian two-hybrid system provides the milieu for the bona fide posttranslational modifications and localizations of most eukaryotic proteins and, therefore, should be a better choice to study proteins of mammalian origin. This chapter depicts the detailed experimental procedures adapted by various laboratories. Researchers with experience in molecular biology could modify the procedures according to their own needs, that is, the choice of restriction sites in the cloning process. The reference list could be of use to researchers who wish to understand more of the system and explore its wider applications.
Keywordsmammalian two hybrid secreted alkaline phosphatase (SEAP) protein-protein interaction PCR cloning
We thank Alina Radziwon, Melissa Ballantine, and Todd Cutts for their contributions to the mammalian two-hybrid analysis. This work was supported by the Research Funding for Public Health Agency of Canada.