Microfluidic integration of Western blotting is enabled by electrotransfer-assisted sodium dodecyl sulfate dilution
Microfluidic integration of Western blotting is enabled by electrotransfer-assisted sodium dodecyl sulfate dilution
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DOI：
10.1039/c2an36033k
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发表时间：
2013-01-01
期刊：
影响因子：
4.2
通讯作者：
Herr, Amy E.
中科院分区：
文献类型：
--
作者：
Hou, Chenlu;Herr, Amy E.
我们将十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）与随后的抗体探针结合在一个单一的单片微设备上，实现微流体Western blotting。芯片上Western blotting成功的一个障碍在于恢复抗体对先前大小（变性、还原）蛋白质的识别。为了克服这一障碍，我们在SDS- page和微室印迹区之间的电转移过程中，利用物种的差异电迁移，局部稀释SDS-蛋白复合物中的游离SDS。SDS的局部稀释减少了SDS与蛋白质的重新结合，为SDS- page后恢复抗体与蛋白质的结合亲和力提供了手段。为了在单个仪器中实现自动化，可编程操作，我们使用1 x 2 mm(2)玻璃微室，该微室具有空间不同，连续的聚丙烯酰胺区域，用于SDS-PAGE，电转移和抗体印迹。SDS-PAGE和电转移条件的优化得到的转移距离为
We integrate sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with subsequent antibody probing in a single, monolithic microdevice to realize microfluidic Western blotting. A hurdle to successful on-chip Western blotting lies in restoring antibody recognition of previously sized (denatured, reduced) proteins. To surmount this hurdle, we locally dilute free SDS from SDS-protein complexes using differential electromigration of the species during electrotransfer between SDS-PAGE and blotting regions of a microchamber. Local dilution of SDS minimizes re-association of SDS with proteins offering means to restore antibody binding affinity to proteins after SDS-PAGE. To achieve automated, programmable operation in a single instrument, we utilize a 1 x 2 mm(2) glass microchamber photopatterned with spatially distinct, contiguous polyacrylamide regions for SDS-PAGE, electrotransfer, and antibody blotting. Optimization of both the SDS-PAGE and electrotransfer conditions yields transfer distances of