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Method for extracting quantitative information relating to interactions between cellular components

机译:提取与细胞成分之间相互作用有关的定量信息的方法

摘要

A method is described to assay for protein interactions in living cells, e.g. by the introduction of two heterologous conjugates into the cell. The method uses the measurement of cellular distribution of a detectable component (e.g. a GFP-labeled˜fluorescent probe) to indicate the presence or absence of an interaction between that component and a second component of interest. The method uses the knowledge that certain components can be stimulated to redistribute within the cell to defined locations. Inducible redistribution systems make it possible to determine if specific interactions occur between components. Inducible systems are described where it is demonstrated that the redistribution stimuli are essentially “null”, in that they affect no other system in the cell during the assay period, other than the component whose redistribution can be induced. Also described is an extraction buffer which is useful in high throughput screening for drugs which affect the intracellular distribution of intracellular components. The extraction buffer comprises a cellular fixation agent and cellular permeabilisation agent. Optimizing the composition of the extraction buffer and its application to various cell types is described.
机译:描述了一种测定活细胞中蛋白质相互作用的方法,例如通过将两种异源结合物引入细胞。该方法使用可检测组分(例如,GFP标记的〜荧光探针)的细胞分布的测量值来指示该组分与所关注的第二组分之间是否存在相互作用。该方法利用以下知识:可以刺激某些成分以在细胞内重新分布到定义的位置。诱导式重新分配系统使确定组件之间是否发生特定的交互成为可能。描述了可诱导系统,其中证明了重分布刺激实质上是“无效”的,因为在测定期间它们不会影响细胞中的其他系统,除了可以诱导其重分布的成分外。还描述了提取缓冲液,其可用于高通量筛选影响细胞内组分的细胞内分布的药物。提取缓冲液包含细胞固定剂和细胞通透剂。描述了提取缓冲液的组成优化及其在各种细胞类型中的应用。

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