Analyses of protein:protein interactions are an integral part of better understanding protein functionality. Mammalian two-hybrid systems provide an excellent means to detect and validate protein interactions within mammalian cells. The next-generation CheckMate™/Flexi® Vector Mammalian Two-Hybrid System provides additional features for ease-of-use and expanded capabilities. The cloning vectors are now compatible with the Flexi® Vector System, allowing easy transfer of protein-coding regions to and from other Flexi® Vectors, which contain specialized functionalities. The reporter vector encodes firefly luciferase with rapid-response characteristics and a hygromycin-resistance marker for generating stable cell lines. In this article, we demonstrate the efficient transfer of protein-coding regions into the CheckMate™/Flexi® Vectors and the ease of screening for interacting partners. We also show how to use the mammalian two-hybrid system to generate stable cell lines and modulate protein interactions with small molecules in a stably transfected cell line.
Promega Notes 94, 12–16.