Matthew B Robers, James D Vasta, Cesear R Corona, Rachel Friedman Ohana, Robin Hurst, Manisha A Jhala, Kenneth M Comess, Keith V Wood
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引用次数: 26
Abstract
Intracellular target affinity and residence time are fundamental aspects of pharmacological mechanism (Lu and Tonge, Curr Opin Chem Biol 14:467-474, 2010). Although various robust biochemical approaches exist to measure these binding characteristics, analysis of compound binding with isolated targets may not accurately reflect engagement in the milieu of living cells. To realize the influence of cellular context, methods are needed that are capable of quantifying affinity and residence time in the presence of the intracellular factors that may impact target engagement. Bioluminescence resonance energy transfer (BRET) offers a solution for intracellular target engagement when quantitative metrics or kinetic analyses are required.
细胞内靶点亲和力和停留时间是药理机制的基本方面(Lu and Tonge, current Opin Chem Biol 14:46 67-474, 2010)。尽管存在各种强大的生化方法来测量这些结合特性，但对化合物与孤立靶标结合的分析可能无法准确反映活细胞环境中的结合。为了实现细胞环境的影响，需要能够量化可能影响目标接合的细胞内因素存在时的亲和力和停留时间的方法。当需要定量测量或动力学分析时，生物发光共振能量转移(BRET)为细胞内目标接合提供了一种解决方案。
期刊介绍：
For over 20 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice.
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