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Abstract for CheckMate™/Flexi® Vector Mammalian
Two-Hybrid
System
Protein interactions are an integral aspect of functional proteomic studies, and
the CheckMate™/Flexi® Vector Mammalian Two-Hybrid System provides a
means 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 to identify modulators of a specific protein:protein
interaction.
The
CheckMate™/Flexi® Vector 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. Association of both domains, driven by the interaction of proteins "X"
and "Y", results in binding to the promoter region and transcriptional
activation of a firefly luciferase reporter gene. Assay of firefly
luciferase activity is sensitive, rapid and easy.
The CheckMate™/Flexi® Vector System relies upon three plasmids that are co-transfected into mammalian cells. 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 GAL4
DNA-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], 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".
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