The CheckMate/Flexi Vector Mammalian Two-Hybrid System provides a means to confirm, validate and study suspected interactions between two proteins or domains and can also be used to generate stable cell lines for cell-based assays. Developed primarily for mammalian proteins of interest, the system can allow protein expression and post-translational modifications in an environment mimicking the native cell milieu. It is patterned on the yeast two-hybrid system with one protein of interest (X) fused to a DNA-binding domain and the other protein (Y) fused to a transcriptional activation domain. The system relies upon three plasmids that are co-transfected into mammalian cells, each plasmid having unique features. The pFN10A (ACT) Flexi Vector contains a herpes simplex virus VP16 transcriptional activation domain upstream of the cloning site, and the pFN11A (BIND) Flexi Vector contains the yeast GAL4DNA-binding domain upstream of the cloning site. The pFN11A (BIND) Flexi Vector also expresses the Renilla reniformis luciferase under the control of the SV40 promoter, allowing normalization for differences in transfection efficiency. The third vector, pGL4.31[luc2P/GAL4UAS/Hygro] Vector, contains five GAL4 binding sites upstream of a minimal TATA box, which is upstream of a firefly luciferase gene that acts as a reporter for interactions between proteins X and Y. This system differs from the original CheckMate Mammalian Two-Hybrid System in that the vectors are compatible with the Flexi Vector System, which allows directional cloning and rapid, efficient and high-fidelity transfer of protein coding regions between a variety of Flexi Vectors.|
Mammalian-Based System: Interactions can be studied in the cell line of choice. Proteins are more likely to be in their native conformation. Post-translational modifications, such as glycosylation, phosphorylation and acylation, are better maintained.Versatile: Vectors are based on the Flexi(R) Cloning technology, enabling convenient transfer of protein-coding regions for additional functional proteomics applications.Convenient: The Dual-Luciferase(R) Reporter Assay System is used for detection.