Santana-Calvo Carmen, Romero Francisco, López-González Ignacio, Nishigaki Takuya
Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (IBT, UNAM), Avenida Universidad 2001, Col. Chamilpa, C.P. 62210, Cuernavaca, Morelos, México.
Biotechniques. 2018 Oct;65(4):211-218. doi: 10.2144/btn-2018-0041.
Fluorescence (or Förster) resonance energy transfer (FRET) is a straightforward and sensitive technique to evaluate molecular interactions. However, most of the popular FRET pairs suffer cross-excitation of the acceptor, which could lead to false positives. To overcome this problem, we selected a large Stokes shift (LSS) fluorophore as a FRET donor. As a successful example, we employed a new FRET pair mAmetrine (an LSS yellow fluorescence protein)/DY-547 (a cyanine derivative) to substitute CFP/fluorescein that we previously employed to study molecular interactions between cyclic nucleotide-binding domains and cyclic nucleotides. The new FRET pair is practically free of cross-excitation of the acceptor. Namely, a change in the fluorescence spectral shape implies evidence of FRET without other control experiments.
荧光（或福斯特）共振能量转移（FRET）是一种评估分子相互作用的直接且灵敏的技术。然而，大多数常用的FRET对存在受体的交叉激发问题，这可能导致假阳性结果。为克服这一问题，我们选择了一种大斯托克斯位移（LSS）荧光团作为FRET供体。作为一个成功的例子，我们采用了新的FRET对，即变色石蒜碱（一种LSS黄色荧光蛋白）/DY-547（一种花青衍生物），来替代我们之前用于研究环核苷酸结合结构域与环核苷酸之间分子相互作用的CFP/荧光素。新的FRET对几乎不存在受体的交叉激发。也就是说，荧光光谱形状的变化意味着FRET的证据，无需其他对照实验。
