Alexa and Oregon Green dyes as fluorescence anisotropy probes for measuring protein-protein and protein-nucleic acid interactions.
Alexa and Oregon Green dyes as fluorescence anisotropy probes for measuring protein-protein and protein-nucleic acid interactions.
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DOI：
10.1016/s0003-2697(02)00325-1
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发表时间：
2002-09
影响因子：
2.9
通讯作者：
E. Rusinova;V. Tretyachenko-Ladokhina;Oana Vele;D. F. Senear;J. B. Alexander Ross
中科院分区：
文献类型：
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作者：
E. Rusinova;V. Tretyachenko-Ladokhina;Oana Vele;D. F. Senear;J. B. Alexander Ross
当Alexa 488、俄勒冈州Green 488和俄勒冈州绿色514（Molecular Probes（尤金，OR））与生物分子偶联时和作为游离于溶液中的模型化合物时，比较它们的荧光性质。我们表明，这些相对较新的，绿色荧光探针是很好的探针的研究热力学的蛋白质-蛋白质和蛋白质-核酸相互作用的荧光各向异性。与荧光素不同，这些染料的发射具有接近中性的最小pH依赖性，并且对光漂白显著不敏感。稳态和时间分辨的荧光各向异性数据进行了比较，两个相互作用的蛋白质的不同大小，并为协会的转录因子与DNA寡核苷酸含有一个特定的结合位点。的温度依赖性的探针的荧光寿命的报告，并讨论了分子的大小和探针运动对稳态各向异性数据的影响。相关时间，荧光寿命，和所观察到的稳态各向异性之间的关键相互作用进行评估。
The fluorescence properties of Alexa 488, Oregon Green 488, and Oregon Green 514 (Molecular Probes (Eugene, OR)) are compared when conjugated to biomolecules and as model compounds free in solution. We show that these relatively new, green fluorescence probes are excellent probes for investigation of the thermodynamics of protein–protein and protein–nucleic acid interactions by fluorescence anisotropy. Unlike fluorescein, the emission of these dyes has minimal pH dependence near neutrality and is significantly less susceptible to photobleaching. Steady-state and time-resolved fluorescence anisotropy data are compared for two interacting proteins of different size and for the association of a transcription factor with a DNA oligonucleotide containing a specific binding site. The temperature dependence of the fluorescence lifetimes of the probes is reported, and the effects of molecular size and probe motion on steady-state anisotropy data are discussed. The critical interplay among correlation time, fluorescence lifetime, and the observed steady-state anisotropy is evaluated.