Protein fractionation in a multicompartment device using Off-Gel™ isoelectric focusing
Protein fractionation in a multicompartment device using Off-Gel™ isoelectric focusing
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DOI：
10.1002/elps.200390030
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发表时间：
2003-01-01
期刊：
影响因子：
2.9
通讯作者：
Rossier, JS
中科院分区：
文献类型：
--
作者：
Michel, PE;Reymond, F;Rossier, JS
提出了一种新的基于离胶等电聚焦（IEF）的蛋白质分级分离技术，该技术在多孔装置中根据蛋白质的等电点（p/）分离蛋白质，其优点是可以直接回收到溶液中用于进一步分析。然后用聚合物纳米电喷雾进行质谱（MS）分析或用生物分析仪进行质量鉴定，对用这种技术获得的蛋白质级分进行表征。这种方法显示了开发替代经典的二维（2-D）凝胶电泳的可能性。对离胶分离过程中的电场分布进行了数值模拟，验证了离胶分离的原理。为了突出该技术的动力学和效率，已经进行了p/蛋白质标记物的实验。此外，对于β-乳球蛋白A和B的分离，分级分离的分辨率显示为0.1 pH单位。此外，在标准增溶缓冲液中进行大肠杆菌提取物的等电分馏，以证明该技术的性能，特别是用于蛋白质组学应用。
A new protein fractionation technique based on off-gel isoelectric focusing (IEF) is presented, where the proteins are separated according to their isoelectric point (p/) in a multiwell device with the advantage to be directly recovered in solution for further analysis. The protein fractions obtained with this technique have then been characterized with polymer nanoelectrospray for mass spectrometry (MS) analyses or with Bioanalyzer for mass identification. This methodology shows the possibility of developing alternatives to the classical two-dimensional (2-D) gel electrophoresis. One species numerical simulation of the electric field distribution during off-gel separation is also presented in order to demonstrate the principle of the purification. Experiments with p/ protein markers have been carried out in order to highlight the kinetics and the efficiency of the technique. Moreover, the resolution of the fractionation was shown to be 0.1 pH unit for the separation of beta-lactoglobulin A and B. In addition, the isoelectric fractionation of an Escherichia coli extract was performed in standard solubilization buffer to demonstrate the performances of the technique, notably for proteomics applications.