Mammalian two-hybrid assay for detecting protein-protein interactions in vivo

Abstract

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.

Fig. 22.1

Principles of the-two hybrid system: DBD and AD are DNA binding and transcriptional activation domains, X and Y are the bait and prey genes

Fig 22.2

SEAP analysis: Chemiluminescence readings of the transfected samples. The depicted constructs were transfected in triplicate, 48 h after transfection, supernatant samples were harvested and subjected to SEAP analysis. The levels of chemiluminescence readings reflect the kinetics of protein-protein interactions