Bailey Michael F, Angley Lauren M, Perugini Matthew A
Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia.
Anal Biochem. 2009 Jul 15;390(2):218-20. doi: 10.1016/j.ab.2009.03.045. Epub 2009 Apr 5.
The fluorescence detection system for the analytical ultracentrifuge (AU-FDS) enables the measurement of hydrodynamic properties and interactions of biomolecules at subnanomolar concentrations. In this study, we describe methods for (i) preparing and purifying fluorescently labeled biomolecules and (ii) determining the meniscus position in the AU-FDS using BODIPY 493/503 fluorescent dye suspended in light oil. We subsequently use these methods to measure the interaction of DNA with Escherichia coli Klenow fragment (KF) and show that KF binds matched DNA to form 1:1 and 2:1 (protein/DNA) complexes with dissociation constants of 4.2 and 22 nM, respectively.
分析超速离心机荧光检测系统（AU-FDS）能够在亚纳摩尔浓度下测量生物分子的流体动力学性质和相互作用。在本研究中，我们描述了以下方法：（i）制备和纯化荧光标记的生物分子；（ii）使用悬浮在轻质油中的BODIPY 493/503荧光染料确定AU-FDS中的弯月面位置。随后，我们使用这些方法测量了DNA与大肠杆菌Klenow片段（KF）的相互作用，结果表明KF与匹配的DNA结合，形成1:1和2:1（蛋白质/DNA）复合物，解离常数分别为4.2和22 nM。
