Rapid, highly efficient extraction and purification of membrane proteins using a microfluidic continuous-flow based aqueous two-phase system
Rapid, highly efficient extraction and purification of membrane proteins using a microfluidic continuous-flow based aqueous two-phase system
复制标题
使用基于微流体连续流的水性两相系统快速、高效地提取和纯化膜蛋白
DOI：
10.1016/j.chroma.2010.10.090
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发表时间：
2011-01-07
影响因子：
4.1
通讯作者：
Liu, Bi-Feng
中科院分区：
文献类型：
--
作者：
Hu, Rui;Feng, Xiaojun;Liu, Bi-Feng
Membrane proteins play essential roles in regulating various fundamental cellular functions. To investigate membrane proteins, extraction and purification are usually prerequisite steps. Here, we demonstrated a microfluidic aqueous PEG/detergent two-phase system for the purification of membrane proteins from crude cell extract, which replaced the conventional discontinuous agitation method with continuous extraction in laminar flows, resulting in significantly increased extraction speed and efficiency. To evaluate this system, different separation and detection methods were used to identify the purified proteins, such as capillary electrophoresis, SDS-PAGE and nano-HPLC-MS/MS. Swiss-Prot database with Mascot search engine was used to search for membrane proteins from random selected bands of SDS-PAGE. Results indicated that efficient purification of membrane proteins can be achieved within 5-7s and approximately 90% of the purified proteins were membrane proteins (the highest extraction efficiency reported up to date), including membrane-associated proteins and integral membrane proteins with multiple transmembrane domains. Compared to conventional approaches, this new method had advantages of greater specific surface area, minimal emulsification, reduced sample consumption and analysis time. We expect the developed method to be potentially useful in membrane protein purifications, facilitating the investigation of membrane proteomics. (C) 2010 Elsevier B.V. All rights reserved.
