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Abstract for CheckMate™ Mammalian Two-Hybrid System
Two-hybrid systems are extremely powerful methods for
detecting protein:protein interactions in vivo. The basis of two-hybrid systems
is the modular domains found in some transcription factors. In the CheckMate™
Mammalian Two-Hybrid System, the pBIND Vector contains the yeast GAL4 DNA-binding
domain upstream of a multiple cloning region, and the pACT Vector contains the
herpes simplex virus VP16 activation domain upstream of a multiple cloning region.
In addition, the pBIND Vector expresses the Renilla reniformis luciferase,
which allows the user to normalize the transfection efficiency. The two genes
encoding the two potentially interactive proteins of interest are cloned into
pBIND and pACT Vectors to generate fusion proteins with the DNA-binding domain
of GAL4 and the activation domain of VP16, respectively. The pG5luc
Vector contains five GAL4 binding sites upstream of a minimal TATA box, which in
turn, is upstream of the firefly luciferase gene (luc+).
The pGAL4 and pVP16 fusion constructs are transfected along with pG5luc
Vector into mammalian cells. Two to three days after transfection, the cells are
lysed, and the amounts of Renilla luciferase and firefly luciferase are
quantitated using the Dual-Luciferase® Reporter Assay System.
Interaction between the two test proteins, as GAL4 and VP16 fusion constructs,
results in an increase in firefly luciferase expression over the negative controls.
Two positive control vectors are provided: pBIND-Id and pACT-MyoD Control Vectors,
containing GAL4:Id and VP16:MyoD, respectively.
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