KRAS4b：用于药物发现的 RAF-1 均相时间分辨荧光共振能量转移测定法。
KRAS4b:RAF-1 Homogenous Time-Resolved Fluorescence Resonance Energy Transfer Assay for Drug Discovery.
机构信息
NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, MD, USA.
出版信息
Methods Mol Biol. 2024;2797:159-175. doi: 10.1007/978-1-0716-3822-4_12.
Homogenous time-resolved FRET (HTRF) assays have become one of the most popular tools for pharmaceutical drug screening efforts over the last two decades. Large Stokes shifts and long fluorescent lifetimes of lanthanide chelates lead to robust signal to noise, as well as decreased false positive rates compared to traditional assay techniques. In this chapter, we describe an HTRF protein-protein interaction (PPI) assay for the KRAS4b G-domain in the GppNHp-bound state and the RAF-1-RBD currently used for drug screens. Application of this assay contributes to the identification of lead compounds targeting the GTP-bound active state of K-RAS.
均相时间分辨荧光共振能量转移（HTRF）测定法在过去二十年中已成为药物筛选的最常用工具之一。镧系螯合物具有较大的Stokes 位移和较长的荧光寿命，与传统的测定技术相比，其信号噪声比更高，假阳性率更低。在本章中，我们描述了用于结合 GppNHp 的 KRAS4b G 结构域和 RAF-1-RBD 的 HTRF 蛋白-蛋白相互作用（PPI）测定法，目前该测定法用于药物筛选。该测定法的应用有助于鉴定针对 K-RAS 的 GTP 结合活性状态的先导化合物。
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