CheckMate? Mammalian Two-Hybrid System

• 产品编号：E2440      品牌：promega       原厂货号：E2440
• 产品分类：蛋白质检测与分析 > 蛋白质相互作用分析 > 报告基因检测
• 应用分类：

其它组分：

描述：

双杂交系统是用于体内蛋白与蛋白相互作用检测的极为有力的工具。此系统的基础是某些转录因子中的模块结构域：即一种能够结合特异DNA 序列的DNA 结合区结构域和一种能够与基础转录元件相互作用的转录激活结构域。与DNA 结合结构域相关的转录激活结构域将促进RNA 聚合酶II 复合体在TATA 盒的组装并增加转录。在CheckMate™ Mammalian Two-Hybrid System (CheckMate™ 哺乳动物双杂交系统中), DNA 结合结构域与转录激活结构域由不同的质粒产生，当一个融合在DNA 结合结构域的蛋白("X") 与第二个融合在转录激活结构域上的蛋白("Y") 相互作用时，二者紧密相关。此系统中，蛋白X 和Y 的相互作用引起报告基因的转录。

原厂资料：

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.

注意事项：

储存于-20℃。