A microchannel solution mixer for studying microsecond protein folding reactions
A microchannel solution mixer for studying microsecond protein folding reactions
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DOI：
10.1063/1.1834698
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发表时间：
2005-01-01
影响因子：
1.6
通讯作者：
Matthews, CR
中科院分区：
文献类型：
--
作者：
Bilsel, O;Kayatekin, C;Matthews, CR
许多蛋白质通过亚毫秒时间范围内的中间体折叠。为了直接监测这些动力学中间体的形成，我们开发了一种简单，耐用，易于组装的连续流动混合器，用于在35-1000 μ s的时间范围内研究折叠反应。该混合器是通过在127 μ m厚的聚酰亚胺或聚醚醚酮聚合物晶片上激光加工75 μ m通道而构造的。对于流速为10-20 mL/min的8 M尿素的1/10稀释反应，可以实现类似于25至类似于50 μ s的混合时间。描述了基于CCD的稳态和基于时间相关单光子计数的荧光检测策略。细胞色素c的重折叠的早期事件的初步结果。(C)2005年美国物理学会。
Many proteins fold through intermediates that are populated in the submillisecond time regime. To monitor directly the formation of these kinetic intermediates, we have developed a simple, robust, easy to assemble continuous flow mixer for studying folding reactions in the 35-1000 mus time regime. The mixer is constructed by laser-machining 75-mum channels in a 127-mum-thick polyimide or polyetheretherketone polymer wafer. Mixing times of similar to25 to similar to50 mus can be achieved for a 1/10 dilution reaction of 8 M urea with flow rates of 10-20 mL/min. CCD-based steady-state and time-correlated single-photon-counting-based fluorescence detection strategies are described. Preliminary results on the early events in the refolding of cytochrome c are presented. (C) 2005 American Institute of Physics.