Fluorescence polarization competition assay: The range of resolvable inhibitor potency is limited by the affinity of the fluorescent ligand
Fluorescence polarization competition assay: The range of resolvable inhibitor potency is limited by the affinity of the fluorescent ligand
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DOI：
10.1177/1087057102239666
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发表时间：
2003-02-01
影响因子：
--
通讯作者：
Huang, XY
中科院分区：
文献类型：
--
作者：
Huang, XY
对于荧光偏振（FP）竞争测定的发展，人们普遍认为，在筛选小分子化合物文库时，应避免紧密结合的荧光配体来鉴定低或中等效力的抑制剂。本文证明，这种说法实际上是一种误解，荧光配体的亲和力越高，可以分辨的抑制剂效力的范围越宽。可以分辨的抑制剂Ki值的下限的近似估计是荧光配体的Kd值。由于FP竞争测定通常在非化学计量滴定条件下进行，因此建议选择具有最高亲和力的荧光配体，其也具有足够的量子产率以满足此类条件。
For the development of fluorescence polarization (FP) competition assays, there is a widespread belief that tight-binding fluorescent ligands should be avoided to identify inhibitors of low or intermediate potency in the screening of small-molecule compound libraries. It is demonstrated herein that this statement is a misconception in fact, the higher the affinity of the fluorescent ligand, the wider the range of inhibitor potency that can be resolved. An approximate estimate for the low end of inhibitor K-i values that can be resolved is the K-d value of the fluorescent ligand. Because FP competition assays are typically conducted under nonstoichiometric titration conditions, it is suggested that a fluorescent ligand of highest affinity that also has an adequate quantum yield to satisfy such conditions be selected.