Multianalyte On-Chip Native Western Blotting
Multianalyte On-Chip Native Western Blotting
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DOI：
10.1021/ac200322z
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发表时间：
2011-05-01
影响因子：
7.4
通讯作者：
Herr, Amy E.
中科院分区：
文献类型：
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作者：
Tia, Samuel Q.;He, Mei;Herr, Amy E.
我们以自动化和统一的微流体格式引入并表征了多重天然蛋白质印迹 (4)。虽然平板凝胶蛋白质印迹法缓慢且费力，但传统的多重印迹法（“重印迹”：用多种抗体探测一个样品）需要更多的资源。在这里，我们详细介绍了三个关键的进步，这些进步使得自动化和快速的微流体替代平板凝胶重印迹成为可能。首先，我们介绍了将针对多个抗体印迹区域的蛋白质印迹与天然聚丙烯酰胺凝胶电泳相结合的测定和微装置设计。这种微流体集成策略克服了传统重印迹通常需要的严格抗体剥离步骤所固有的非特异性材料损失；所述条件会严重限制分析物定量。其次，为了告知多重微流体装置的合理设计，我们开发了一种用于印迹区域上分析物捕获的分析模型。与经验观察结果进行比较，捕获效率 > 85%。第三，我们引入了无标记检测，使得同时定量多重测量成为可能，而无需对样品进行预标记。检测线性动态范围涵盖 8-800 nM，检测可在 5 分钟内完成。由于速度、自动化、增强的定量能力以及传统平板凝胶蛋白质印迹的难度，微流体多重天然蛋白质印迹应该在系统生物学中得到应用，特别是在蛋白质亚型和多聚体蛋白质复合物的分析中。
We introduce and characterize multiplexed native Western blotting (4) in an automated and unified microfluidic format. While slab gel Western blotting is slow and laborious, conventional multiplexed blotting ("reblotting": probing one sample with multiple antibodies) requires even more resources. Here we detail three key advances that enable an automated and rapid microfluidic alternative to slab gel reblotting. First, we introduce both assay and microdevice designs that integrate protein blotting against multiple antibody blotting regions with native polyacrylamide gel electrophoresis. This microfluidic integration strategy overcomes nonspecific material losses inherent to harsh antibody stripping steps typically needed for conventional reblotting; said conditions can severely limit analyte quantitation. Second, to inform rational design of the multiplexed microfluidic device we develop an analytical model for analyte capture on the blotting regions. Comparison to empirical observations is reported, with capture efficiencies of > 85%. Third, we introduce label free detection that makes simultaneous and quantitative multiplexed measurements possible without the need for prelabeling of sample. Assay linear dynamic range spans 8-800 nM with assay completion in 5 min. Owing to the speed, automation, enhanced quantitation capability, and the difficulty of conventional slab gel Western reblotting, microfluidic multiplexed native Western blotting should find use in systems biology, in particular in analyses of protein isoforms and multimeric protein complexes.