Department of Chemistry, University of Michigan, 930 N. University Ave., Ann Arbor, Michigan 48109, USA.
LI-COR Biosciences, 4647 Superior St., Lincoln, Nebraska 68504, USA.
J Chromatogr A. 2022 Aug 30;1679:463389. doi: 10.1016/j.chroma.2022.463389. Epub 2022 Jul 30.
Traditional Western blots are commonly used to separate and assay proteins; however, they have limitations including a long, cumbersome process and large sample requirements. Here, we describe a system for Western blotting where capillary gel electrophoresis is used to separate sodium dodecyl sulfate-protein complexes. The capillary outlet is threaded into a piezoelectric inkjetting head that deposits the separated proteins in a quasi-continuous stream of <100 pL droplets onto a moving membrane. Through separations at 400 V/cm and protein capture on a membrane moving at 2 mm/min, we are able to detect actin with a limit of detection at 8 pM, or an estimated 5 fg injected. Separation and membrane capture of sample containing 10 proteins ranging in molecular weights from 11 - 250 kDa was achieved in 15 min. The system was demonstrated with Western blots for actin, β-tubulin, ERK1/2, and STAT3 in human A431 epidermoid carcinoma cell lysate.
传统的 Western blot 常用于分离和检测蛋白质；然而，它们存在一些局限性，包括冗长而繁琐的过程和大量的样品需求。在这里，我们描述了一种 Western blot 系统，其中毛细管凝胶电泳用于分离十二烷基硫酸钠-蛋白质复合物。毛细管出口被插入到压电喷墨打印头中，将分离的蛋白质以 100 pL 以下的近乎连续的液滴流沉积到移动的膜上。通过在 400 V/cm 下进行分离，以及以 2 mm/min 的速度移动的膜上的蛋白质捕获，我们能够以 8 pM 的检测限检测肌动蛋白，或估计注入的 5 fg。在 15 分钟内实现了 10 种分子量为 11-250 kDa 的蛋白质的分离和膜捕获。该系统通过人 A431 表皮样癌细胞裂解物中的肌动蛋白、β-微管蛋白、ERK1/2 和 STAT3 的 Western blot 进行了演示。
