The CheckMate™/Flexi® System is designed to confirm, validate and study suspected interactions between two proteins or domains. This system can also be used to generate stable cell lines for cell-based assays. The system can allow mammalian protein expression and post-translational modification studies in an environment mimicking the native cell milieu. The CheckMate™/Flexi® System 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.
The plasmids are available and can be ordered separately from the system.
This system differs from the original CheckMate™ Mammalian Two-Hybrid System in that the vectors are compatible with the Flexi® Vector System, enabling directional cloning and rapid, efficient and high-fidelity transfer of protein coding regions between a variety of Flexi® Vectors.
- Confirm, validate and study suspected interactions between two proteins or domains.
GenBank® Accession Number and Sequence Information