Dual-Channel Stopped-Flow Apparatus for Simultaneous Fluorescence, Anisotropy, and FRET Kinetic Data Acquisition for Binary and Ternary Biological Complexes.
Dual-Channel Stopped-Flow Apparatus for Simultaneous Fluorescence, Anisotropy, and FRET Kinetic Data Acquisition for Binary and Ternary Biological Complexes.
复制标题
DOI：
10.3390/bios10110180
复制
发表时间：
2020-11-19
期刊：
影响因子：
--
通讯作者：
Parkhurst LJ
中科院分区：
文献类型：
--
作者：
Delgadillo RF;Carnes KA;Valles-Villarreal N;Olmos O;Zaleta-Rivera K;Parkhurst LJ
停流装置 (SF) 通过在亚毫秒范围内混合反应物来跟踪分子事件。可以通过记录纳摩尔浓度的荧光、F(t)、各向异性、r(t)、偏振、p(t) 或 FRET、F(t)FRET 迹线来监测内在或外在标记的生物分子的反应。这些动力学测量对于阐明反应机制、结构信息甚至热力学至关重要。在单探测器 SF 或 L 配置中，通过切换发射偏振器的方向以收集 IVV 和 IVH 信号来获取 r(t)、p(t) 和 F(t) 迹线，但这需要两次实验。在双探测器 SF 或 T 型配置中，这些痕迹是在单次实验中收集的，但这增加了设备的复杂性和价格。在此，我们提出了一种单探测器双通道SF，可同时获得F(t)和r(t)迹线，其中光弹性调制器以50 kHz频率使激发光平面振荡90°，发射信号由一组电子滤波器处理，将其分成r(t)和F(t)模拟信号，这些信号被数字化并存储在单独的电子表格中 通过定制的仪器控制软件。我们评估了用我们的双通道 SF 和传统方法获得的二元和三元生物复合物的缔合动力学；例如在魔角处使用单个偏振器获取 F(t)，一组偏振器跟踪 F(t) 和 r(t)，以及通过能量转移猝灭实现 F(t)FRET。我们的双通道 SF 节省了标记材料，并且产生的速率常数与传统方法非常一致。
The Stopped-Flow apparatus (SF) tracks molecular events by mixing the reactants in sub-millisecond regimes. The reaction of intrinsically or extrinsically labeled biomolecules can be monitored by recording the fluorescence, F(t), anisotropy, r(t), polarization, p(t), or FRET, F(t)FRET, traces at nanomolar concentrations. These kinetic measurements are critical to elucidate reaction mechanisms, structural information, and even thermodynamics. In a single detector SF, or L-configuration, the r(t), p(t), and F(t) traces are acquired by switching the orientation of the emission polarizer to collect the IVV and IVH signals however it requires two-shot experiments. In a two-detector SF, or T-configuration, these traces are collected in a single-shot experiment, but it increases the apparatus’ complexity and price. Herein, we present a single-detector dual-channel SF to obtain the F(t) and r(t) traces simultaneously, in which a photo-elastic modulator oscillates by 90° the excitation light plane at a 50 kHz frequency, and the emission signal is processed by a set of electronic filters that split it into the r(t) and F(t) analog signals that are digitized and stored into separated spreadsheets by a custom-tailored instrument control software. We evaluated the association kinetics of binary and ternary biological complexes acquired with our dual-channel SF and the traditional methods; such as a single polarizer at the magic angle to acquire F(t), a set of polarizers to track F(t), and r(t), and by energy transfer quenching, F(t)FRET. Our dual-channel SF economized labeled material and yielded rate constants in excellent agreement with the traditional methods.
登录
查看更多内容
影响因子：
2.9
作者：
Delgadillo, Roberto F.;Whittington, JoDell E.;Parkhurst, Lawrence J.
通讯作者：
Parkhurst, Lawrence J.
影响因子：
4.4
作者：
Eyring, H
通讯作者：
Eyring, H
影响因子：
2.9
作者：
Galletto, R;Maillard, R;Bujalowski, W
通讯作者：
Bujalowski, W
影响因子：
1.6
作者：
BERGER, RL;BALKO, B;FRIAUF, W
通讯作者：
FRIAUF, W
DOI：
10.1016/s0016-0032(40)90963-2
发表时间：
1940-01-01
期刊：
JOURNAL OF THE FRANKLIN INSTITUTE
影响因子：
--
作者：
Chance, B
通讯作者：
Chance, B