Membrane-assisted online renaturation for automated microfluidic lectin blotting.
Membrane-assisted online renaturation for automated microfluidic lectin blotting.
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DOI：
10.1021/ja207963f
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发表时间：
2011-12-14
影响因子：
15
通讯作者：
Herr, Amy E.
中科院分区：
文献类型：
--
作者：
He, Mei;Novak, Jan;Julian, Bruce A.;Herr, Amy E.
异常糖基化在包括癌症在内的多种疾病中起着关键作用。介绍了一种微流控凝集素印迹平台，以实现和加快基于蛋白质大小和对特定凝集素的亲和力的蛋白质糖基化的鉴定。集成的多阶段分析消除了板状凝胶凝集素印迹所需的手动干预步骤，增加了总分析通量，限制了试剂和样品消耗，并使用一台仪器完成。该试验包括非还原十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE），然后进行在线尺寸后SDS过滤和基于凝集素的亲和印迹。重要的功能是通过设备和测定的进步，使集成的纳米多孔膜侧翼的中央微室，以创建亚纳升体积的隔室，捕获SDS-蛋白质复合物，并允许电泳SDS去除缓冲液交换赋予。通过SDS处理的绿色荧光蛋白（GFP）的定量评估，优化了凝集素的蛋白结合的重现。从约3 μL样品中在约6分钟内探测到含有半乳糖缺陷型O-聚糖的异常糖基化IgA 1。这种新的微流控凝集素印迹法提供了一种快速和自动化的测定异常糖基化的评估。
Aberrant glycosylation plays a pivotal role in a diverse set of diseases including cancer. A microfluidic lectin blotting platform is introduced to enable and expedite the identification of protein glycosylation based on protein size and affinity for specific lectins. The integrated multi-stage assay eliminates manual intervention steps required for slabgel lectin blotting, increases total assay throughput, limits reagent and sample consumption, and completes using one instrument. The assay is comprised of non-reducing sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by on-line post-sizing SDS filtration and lectin-based affinity blotting. Important functionality is conferred through both device and assay advances that enable integration of nanoporous membranes flanking a central microchamber to create sub-nanoliter volume compartments which trap SDS-protein complexes and allow electrophoretic SDS removal with buffer exchange. Recapitulation of protein binding for lectin was optimized through quantitative assessment of SDS-treated green fluorescent protein (GFP). Aberrantly glycosylated IgA1 with galactose-deficient O-glycans was probed in ~6 min from ~3 μL of sample. This new microfluidic lectin blotting provides a rapid and automated assay for assessment of aberrant glycosylation.
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影响因子：
3.2
作者：
Propheter, Daniel C.;Hsu, Ku-Lung;Mahal, Lara K.
通讯作者：
Mahal, Lara K.
影响因子：
7.4
作者：
Tia, Samuel Q.;He, Mei;Herr, Amy E.
通讯作者：
Herr, Amy E.
影响因子：
3.3
作者：
Bales, BL;Messina, L;Nascimento, OR
通讯作者：
Nascimento, OR
影响因子：
2.9
作者：
Enoki, S;Saeki, K;Kuwajima, K
通讯作者：
Kuwajima, K
影响因子：
46.9
作者：
Pédelacq, JD;Cabantous, S;Waldo, GS
通讯作者：
Waldo, GS